PCR Protocol for Fish and Seafood Authentication - MSc Thesis

The Optimization and Validation of a Polymerase Chain Reaction Protocol

for Fish and Seafood Authenticity based on the Cytochrome b Gene

M.Sc. thesis submitted by:

D w i y i t n o

Catholic University of Applied Science (KaHo) Sint Lieven, Belgium Dublin Institute of Technology, Ireland

Universidade Católica Portuguesa, Portugal Anhalt University of Applied Sciences, Germany

2008

ERASMUS MUNDUS MASTER COURSE

SEFOTECH.NUT

The Optimization and Validation of a Polymerase Chain Reaction Protocol

for Fish and Seafood Authenticity based on the Cytochrome b Gene

M.Sc. thesis submitted by:

D w i y i t n o Project Coordinator: Prof. Dr. Chris Van Keer Supervisors: Prof. Dr. Chris Van Keer Dr. Koen Parmentier Co-supervisor: Stefan Hoffman, M.Sc.

February 2008

KaHo-Sint Lieven School for Engineering Gebr. Desmetstraat 1 9000 Gent - Belgium

The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene

Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) i

ABSTRACT

Dwiyitno. The optimization and validation of a polymerase chain reaction protocol for fish and seafood authenticity based on the cytochrome b gene. (Under direction of Prof. Dr. Chris Van Keer and Dr. Koen Parmentier; supervised by Stefan Hoffman, M.Sc)

Cytochrome b mtDNA has been widely applied for identification of fish and seafood,

either in fresh or processed products. The successful application of product authentication based on genomic profiling considerably depends on the primer design which is used to amplify the targeted DNA fragment. Several primers have been developed specifically to identify particular groups of fish, crustaceans and molluscs. However, universal primers for identification of most fish, crustaceans, and molluscs based on the cyt b region have not been established yet. This study focused on the development of universal primers for fish and seafood authenticity based on the cyt b gene. Universal primers are essential, particularly for identification of unrecognizable samples such as fish fillet, surimi and mixed products. In addition, since DNA quality plays an important contribution on PCR amplification, investigation on the different DNA isolation methods was carried out.

Firstly, CytBL1 and CytBH primers which have successfully been used to amplify ~357bp of cyt b gene on various fish were optimized to amplify selected fish, crustaceans and molluscs. Secondly, since this primer couple was not optimum for crustacean, mollusc, and some fishes, degenerate primers were designed by introducing wobbles. Notably, other degenerate primers were evaluated to amplify ~410bp of expected fragment. Evaluation of 3 classical DNA extraction methods and a commercial kit was studied to isolate total DNA of selected samples.

The results showed that the CytBL1 and CytBH failed to amplify crustacean and mollusc. Likewise, some species of fish failed to be amplified by this primer couple. The degenerate primers (CytBL1C and CytBHW) are promising to be employed universally for species identification of crustaceans and molluscs. Other universal primers (UCYTB151BF/UCYTB271R and UCYTB152BF/UCYTB271R) effectively amplified all fish, crustaceans, and molluscs tested in this study and thereby can be considered as the first universal primers applicable for fish and seafood. Sequence analysis proved that all validated primers effectively generate 356-358bp and 398-411bp of cyt b region. The similarity index of PCR products against the libraries varied between 92% and 100%. RT-PCR was applicable to differentiate between selected samples based on their melting points (Tm). In comparison to the classical methods, the commercial kit offers simplicity procedure and yielded the better quality of DNA isolate for PCR purposes. Key words: authenticity, mitochondrial cytochrome b, PCR primers, fish and seafood,

DNA sequencing


Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) ii

ACKNOWLEDGEMENTS

This thesis was a part of my master course in Food Science, Technology, and

Nutrition (SEFOTECHnut). It was funded by the European Commission under the

Erasmus Mundus framework. The studies were undertaken in 2006-2008 at Catholic

University of Applied Science (KaHo) Sint Lieven-Belgium as the host university and

partially at Dublin Institute of Technology-Ireland, Universidade Cathólica Portuguesa-

Portugal, and Anhalt University of Applied Sciences-Germany.

Many people took part in my M.Sc studies and without them this thesis would not

exist. I am deeply grateful to both of my advisors, Prof. Dr. Chris Van Keer (KaHo Sint

Lieven) and Dr. Koen Parmentier (ILVO), who have encouraged and mentored me during

this works. Prof. Chris is also the coordinator of SEFOTECHnut, thank you for giving me

opportunity to be part of this master course. This thesis project was carried out at Institute

for Agriculture and Fisheries Research (ILVO), Belgium. I would like to express my

gratitude to Stefan Hoffman M.Sc and his research group (Daphne and Sabrine) for the

invaluable support and supervising me during this research and writing the report. They

have introduced me to many basic theories and practical application in biomolecular work.

I gratefully acknowledge Dr. Kris Cooreman, the Director of ILVO-Fisheries Department,

for the opportunity to work at his laboratory. Thank all colleagues in Ankerstraat 1 for

creating so enjoyable atmosphere to work in. I also thank my reviewers, Prof. Dr. Dirk

Iserentant and Prof. Jan Song (Gent University), for evaluating the manuscript and their

comments during my public defense.

I wish to thank my present employer at Research Center for Marine and Fisheries

Product Processing and Biotechnology-Jakarta (Prof. Dr. Hari Eko Irianto, Prof. Dr.

Sumpeno Putro, Prof. Dr. Endang Sri Heruwati, and Dr. Singgih Wibowo, M.S.), my

former employer (Dr. Ahmad Dimyati, M.S. and Dr. W. Farid Ma’ruf, M.Sc) and all of my

workmates, for their support. My warmest thanks belong to my parents, my parents in law,

Om Samso-Bulik Murni, Wa Ann and her family in De Pinte, for all the support and

understanding. Finally, I deeply thank my beloved wife, Lusi, and my juniors (Rizan and

Fitri) for all their patience and comfort during these years.

Gent-Oostende, February 2008

Dwiyitno


Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) iii

TABLE OF CONTENTS

ABSTRACT ..................................................................................................................... i

ACKNOWLEDGEMENTS ............................................................................................. ii

TABLE OF CONTENTS ................................................................................................. iii

LIST OF FIGURES .......................................................................................................... v

LIST OF TABLES ........................................................................................................... vii

I. INTRODUCTION .......................................................................................................... 1

II. REVIEW OF LITERATURE ...................................................................................... 3 2.1. The importance of product authenticity ................................................................ 3 2.2. Analytical methods for product identification ...................................................... 4 2.2.1. Traditional approaches ............................................................................... 5 2.2.2. Protein based methods (Proteomics) .......................................................... 5 2.2.3. DNA based methods (Genomics) ............................................................... 6 2.2.4. Other methods ............................................................................................ 8 2.3. Genomic identification based on mitochondrial DNA ......................................... 8 2.4. Fish and seafood authenticity based on the cytochrome b gene ........................... 11 2.4.1. Specimen and treatment of sample ............................................................. 11 2.4.2. Isolation of DNA ........................................................................................ 12 2.4.2.1. Tissue digestion ............................................................................. 13 2.4.2.2. Separating proteins and contaminants ........................................... 14 2.4.2.3. Precipitation and recovery of DNA ............................................... 15 2.4.2.4. Commercial kits ............................................................................. 16 2.4.3. Determination of DNA yield and purity ..................................................... 17 2.4.4. Polymerase chain reaction (PCR) ............................................................... 19 2.4.4.1. Primer design ................................................................................. 20 2.4.4.2. Components of PCR reaction ........................................................ 22 2.4.5. PCR product analysis ................................................................................. 23 2.4.5.1. DNA sequencing .......................................................................... 24 2.4.5.2. Fingerprinting techniques .............................................................. 25 2.4.5.3. Other techniques ............................................................................ 27

III. OBJECTIVES OF PRESENT STUDY ...................................................................... 28 3.1. Problems .............................................................................................................. 28 3.2. Objectives ............................................................................................................ 28 3.3. Research framework ............................................................................................ 29

IV. MATERIALS AND METHODS ............................................................................... 30 4.1. Construction of reference database of cyt b genes .............................................. 30 4.2. Sample collection and preservation ..................................................................... 30 4.3. Comparison of DNA extraction methods ............................................................ 31 4.3.1. DNA extraction method 1 (Promega) ......................................................... 32 4.3.2. DNA extraction method 2 (Hsieh et al., 2005)............................................ 32 4.3.3. DNA extraction method 3 (Wasko et al., 2003) .......................................... 32 4.3.4. DNA extraction method 4 (Asahida et al., 1996) ........................................ 33


Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) iv

4.3.5. Determination of DNA yield ..................................................................... 33 4.3.5.1. Standard curve .............................................................................. 34 4.3.5.2. Sample Analysis ........................................................................... 35 4.3.6. Evaluation of DNA purity ......................................................................... 35 4.3.7. PCR assay .................................................................................................. 36 4.3.8. Visualization of PCR product .................................................................... 37 4.4. Optimization and validation of a universal PCR protocol ................................... 37 4.4.1. Primer design ............................................................................................. 37 4.4.2. PCR assay .................................................................................................. 38 4.4.3. Direct sequencing of PCR product ............................................................ 38 4.4.4. Real Time PCR application ....................................................................... 39 4.5. Data analysis ........................................................................................................ 40

V. RESULTS .................................................................................................................... 41 5.1. Comparison of DNA extraction methods ............................................................ 41 5.2. Evaluation and optimization of CytBL1 and CytBH primers ............................. 42 5.3. Validation of degenerate primers based on CytBL1 and CytBH ......................... 44 5.4. Optimization of universal primers based on UCYTB151F and UCYTB270R .... 45

5.4.1. Validation of UCYTB151BF/UCYTB271R primers ................................ 46 5.4.2. Validation of UCYTB152BF/UCYTB271R primers ................................ 48 5.5. PCR product analysis .......................................................................................... 50 5.5.1. Direct sequencing ...................................................................................... 50 5.5.2. Melting temperature profiles ..................................................................... 51

VI. DISCUSSION ............................................................................................................ 53 6.1. Comparison of DNA extraction methods ............................................................ 53 6.2. Optimization and validation of universal primers ............................................... 54 6.3. PCR product analysis .......................................................................................... 56

VII. CONCLUSIONS ....................................................................................................... 58

REFERENCES ................................................................................................................. 59

LIST OF ABBREVIATIONS ........................................................................................... 68

LIST OF WEBSITES ....................................................................................................... 60

APPENDIXES .................................................................................................................. 70


Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) v

LIST OF FIGURES

II. REVIEW OF LITERATURE

Figure 2-1. The application of traceability in fish and seafood product ................................. 4

Figure 2-2. Link between different identification techniques and their tools ........................ 7

Figure 2-3. Illustration of mitochondria in a cell and its genomic map ................................. 10

Figure 2-4. DNA spectra presented by spectrophotometer and spectrofluorometer ................ 18

Figure 2-5. The principle of PCR amplification ..................................................................... 19

III. OBJECTIVES OF PRESENT STUDY ............................................................................ 28

Figure 3-1. The framework of the study ................................................................................. 29

IV. MATERIALS AND METHODS

Figure 4-1. Genomic database obtained from NCBI and FishTrace ...................................... 30

Figure 4-2. An example of standard curve for spectrofluorometric measurement .................. 34

Figure 4-3. The application of NanoDrop and its typical data output ..................................... 35

Figure 4-4. Apparatus for PCR application ............................................................................. 36

Figure 4-5. The scheme of targeted fragments generated by the evaluated primers ............... 38

Figure 4-6. Software for sequence chromatogram analysis: ChromasPro and BioEdit .......... 39

Figure 4-7. Typical outputs of RT-PCR: annealing curve and melting curve ......................... 40

Figure 4-8. An example of BLAST analysis obtained via GenBank and FishTrace................ 40

V. RESULTS

Figure 5-1. Electrophoresis profile of DNA isolates .............................................................. 42

Figure 5-2. Electrophoresis profile of PCR products following different extraction methods ................................................................................................................................ 42

Figure 5-3. Electrophoresis profile of PCR products with CytBL1/CytBH on fish ................. 43

Figure 5-4. Electrophoresis profile of PCR products with CytBL1/CytBH on crustaceans and molluscs ................................................................................. 43

Figure 5-5. Electrophoresis profile of PCR products at different concentrations of MgCl2 ... 43

Figure 5-6. Electrophoresis profile of PCR products produced by degenerate primers based on CuyBL1/CytBH on selected samples ................................................... 44

Figure 5-7. Electrophoresis profile of PCR products with CytBL1C/CytBHW on fish .......... 44

Figure 5-8. Electrophoresis profile of PCR products with CytBL1C/CytBHW on crustaceans ................................................................................................................. 45

Figure 5-9. Electrophoresis profile of PCR products with CytBL1C/CytBHW on molluscs 45


Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) vi

Figure 5-10. Electrophoresis profile of PCR products produced by UCYTB151BF on selected samples ............................................................................................ 45

Figure 5-11. Electrophoresis profile of PCR products produced by UCYTB152BF on selected samples ............................................................................................ 46

Figure 5-12. Electrophoresis profile of PCR products with UCYTB151BF/UCYTB271R on fish-A ............................................................................................................. 46

Figure 5-13. Electrophoresis profile of PCR products with UCYTB151BF/UCYTB271R on fish-B ....................................................................................................................... 47 Figure 5-14. Electrophoresis profile of PCR products with UCYTB151BF/UCYTB271R on crustaceans ............................................................................................................. 47 Figure 5-15. Electrophoresis profile of PCR products with UCYTB151BF/UCYTB271R on molluscs .................................................................................................................. 47

Figure 5-16. Electrophoresis profile of PCR products with UCYTB152BF/UCYTB271R on fish-A ...................................................................................................................... 48

Figure 5-17. Electrophoresis profile of PCR products with UCYTB152BF/UCYTB271R on fish-B ....................................................................................................................... 48

Figure 5-18. Electrophoresis profile of PCR products with UCYTB152BF/UCYTB271R on crustaceans ............................................................................................................. 49

Figure 5-19. Electrophoresis profile of PCR products with UCYTB152BF/UCYTB271R on molluscs .................................................................................................................. 49

Figure 5-20. Variations of melting peak on selected samples ................................................. 52

VI. DISCUSSION

Figure 6-1. Multiple alignment of evaluated primers with cyt b gene of selected references.. 55 APPENDIXES

Figure I. List of samples used in this study ......................................................................... 70

Figure III. Electrophoresis profile of gradient tests ............................................................... 72

Figure IV. Sequence chromatogram profiles of PCR products ............................................. 74


Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) vii

LIST OF TABLES

II. REVIEW OF LITERATURE

Table 2-1. Applicable categorical levels of each molecular marker or gene region ............... 10

Table 2-2. Various primer couples used to amplify cyt b region of fish and seafood ............. 21

IV. MATERIALS AND METHODS

Table 4-1. Protocol used to prepare the standard curve ......................................................... 34

Table 4-2. Composition of PCR reaction ............................................................................... 36

Table 4-3. Set of primers used in this study ........................................................................... 37

Table 4-4. PCR condition of the different primer sets ........................................................... 38

Table 4-5. Reaction components of RT-PCR amplification ................................................... 39

Table 4-6. The thermal profile of RT-PCR amplification ...................................................... 40

V. RESULTS

Table 5-1. DNA concentration and purity yielded by different extraction methods ............... 41

Table 5-2. Optimal annealing temperature (ºC) of validated primer couples .......................... 49

Table 5-3. Sequence analysis of selected samples .................................................................. 51

Table 5-4. Variations of melting point and GC content of selected samples .......................... 52 APPENDIXES

Table II. List of chemicals, solution and equipment ............................................................ 71


Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) 1

CHAPTER I INTRODUCTION

Authenticity of fish and seafood products is important to implement the labeling

regulation as set by many countries. The EU, for example, regulates traceability of feed

and food ingredients and food sources to protect consumers from fraud and adulteration.

Identification of product authenticity is essential to prevent substitution of commercially

important products with less valuable species (Trotta et al., 2005). In addition, authenticity

is advantageous for either food producers or food inspectors to assure the species included

in the formulations of the products (Pineiro et al., 1999). Recently, traceability is also

becoming a concern in Canada, Japan, Australia, and New Zealand (Loftus, 2005; Food

Quality News, 2006).

Traditionally, identification of fish is carried out based on the available external

characteristics. With concern to the prospect of growing international trade and an

increasing number of potentially marketable species, it is worthwhile to conduct rapid and

accurate analytical methods to distinguish fish and seafood products without using

morphological features. Protein electrophoresis and molecular biological methods are

valuable methodologies for identification of fish and seafood authenticity (Bossier, 1999;

Etienne et al., 1999; Rehbein et al., 1999; Pineiro et al., 1999; Bossier & Cooreman,

2000).

Protein electrophoresis is an earlier method developed using either SDS-PAGE,

agarose GE or IEF patterns of water soluble sarcoplasmatic protein. Nevertheless, there are

disadvantages regarding these identification methods. As protein is denatured or altered in

consequent of processing, especially by heating, protein based techniques are only

applicable to fresh/raw products, frozen fillets and mildly treated fish and seafood (Wolf et

al., 2000). In addition, the resulted patterns from these methods can be different depending

on the individual age, length, development stage, or environment condition which in turn

leads to a high degree of intra species variation.

The most prominent techniques for species identification are based on the analysis of

nucleic acids, either nuclear or mitochondrial DNA (Pineiro et al., 1999). The significant

advantage of DNA based methods is their ability to identify not only fresh and frozen, but

also processed, degraded and mixed products. In general, DNA based techniques rely on

polymerase chain reaction (PCR) and PCR product analysis. Fingerprinting techniques, for



instance RFLP, AFLP, RAPD, SSCP, and DGGE/TGGE, have been demonstrated as fast,

cheap and straightforward gene identification (Mackie et al., 2000; Hird et al., 2005).

However, sequencing is known as the most accurate technique to produce a high resolution

genomic identity among examined species (Bartlett & Davidson, 1991; Unseld et al.,

1995).

Mitochondrial DNA (mtDNA) has been used world wide for species identification,

including fish and seafood products. Since mtDNA is maternally inherited, the use of this

DNA may reduce the taxonomic uncertainty caused by hybridization which exists in

nDNA (Ward et al., 2005). Specifically, DNA barcodes focus on the 600 bp sequence of

cytochrome oxidase I (COI), while the EU has been developing genomic data base for

commercially important fish based on mitochondrial cytochrome b (cyt b) and nuclear

rhodopsin (Ratnasingham & Hebert, 2007; www.fishtrace.org).

Cyt b gene has been widely applied to identify various species of fish and seafood.

Several protocols have been developed specifically to identify particular groups of fish and

seafood, including tunas/family Scombroidae (Bartlett & Davidson, 1991; Rehbein et al.,

1997), caviar (DeSalle & Birstein, 1996), salmonoid (Rehbein et al., 1997), flatfish

(Céspedes et al., 1998), mollusc and arthropods (Meritt et al., 1998), puffer fish (Hsieh et

al., 2003), gadoid/family Gadidae (Aranishi et al., 2005), billfish/family Istiophoridae

(Hsieh et al., 2005; Richardson et al., 2006) and the most recently teleost fish (Sevilla et

al., 2007). However, a universal protocol, particularly universal primers based on cyt b

gene for identification of the majority of fish, crustaceans, and molluscs, has not been

established yet.



CHAPTER II REVIEW OF LITERATURE

2.1. The importance of product authenticity

Traceability is increasingly recognized as a standard management tool across the

agri-food industry, mainly due to recent trends on food crises as well as the consequent

demands for global transparency within the food chain (Loftus, 2005). Studies in the U.S.

fish market showed that due to the highly demand and popularity, at least 77% of red

snapper were commonly mislabeled or substituted with the less valuable species (Marco et

al., 2004). Similarly, the expensive category of grouper (genera Epinephelus and

Mycoteroperca) are frequently misidentified as nile perch (Lates niloticus) or the wreck

fish (Polyprion americanus), which is closely related and can not be identified when their

morphological features disappeared (Trotta et al., 2005). This mislabeling, misbranding,

and economic adulteration are illegal practices and consequently to gain financially

consumer fraud.

From the viewpoint of food safety, authenticity could protect public from poisoning

incidents due to ingestion of toxic products. Quality assurance has become the top priority

of meat retailers and producers due to the Bovine Spongiform Encephalopathy (BSE)

crisis. BSE almost caused the collapse of beef industry in England and the rest of Europe,

when their beef was banned in many countries (Hanluain, 2001; Necidová et al., 2002).

Serious food poisoning incidents due to ingestion of toxic puffer fish or toxic dried fish

fillets have been reported in Taiwan (Hwang et al., 1995; Hsieh et al., 2002, Cheng et al.,

2003). This is due to the difficulty for manufacturers and consumers to distinguish

morphological similarities between Lagocephalus lunaris and L. gloveri. Additionally,

Takifugu oblongus is often abused as the material for dried fish fillet. With respect to the

fact that awareness on GMO’s food arises all over the world, it becomes essential to

establish consumer protection from GMO jeopardy (Reg. No.1829/2003).

For traceability along the food (supply) chain, particularly for meat and meat

products, several aspects, such as information of animal species, origin, age, composition

and production system are important (Schwagele, 2005). Regulation (EC) 2065/2001 for

example, emphasizes that traceability of fish product requires information concerning

species identity, geographical origin, and method of production (wild or farmed, organic or

intensive). Figure 2-1 shows the illustration of DNA application on the traceability of



seafood product. Hence, for large scale biodiversity monitoring purposes, it is worthwhile

to develop a rapid and accurate identification tool. Interestingly, deoxyribonucleic acid

(DNA) based technology can overcome the difficulties of tracing animal identity and

animal by-products shown by conventional tagging and labelling systems (Hayes et al.,

2005).

Figure 2-1. The application of traceability in fish and seafood product

(Re-illustrated based on Loftus & Laronde, 2005; Thompson et al., 2005)

2.2. Analytical methods for product identification

A key feature of any traceability system has to be able to clearly identify that to be

traced. Ideally, the product identifier should: (a) uniquely identify the unit or batch, (b)

retain identity throughout the product life-cycle, (c) not hinder its host, and should be (d)

secured (fraud proof) and (e) permanent, (f) simple to read and capture identifying data

(Loftus, 2005). Accordingly, there is no single identification system likely to meet all of

these requirements.

Generally, two types of product identifier can be distinguished, i.e. external

identifiers and internal/biometric identifiers that are an integral part of the animal or

product. External types include either manual methods such as paper labels, ink brands

(tattoos), and ear tags, or electronic methods such as Radio Frequency Identification

(RFID) tags and injectable microchips (Loftus & Laronde, 2005; Thompson et al., 2005).

The advantage of these approaches is their ability to encode different types of information

and the relative ease of reading the data, particularly from electronic identifiers. On the

other hand, biometric labelling systems incorporate biological data and therefore cannot

easily be faked, altered or appropriated. The technologies include DNA profiling, retinal

scanning and nose printing (Loftus & Laronde, 2005).

RFID movement monitoring (Growing stage)

Barcode label tracking (Processing & distribution)

DNA identity DNA identity Harvest sampling

RFID & Hatchery sampling

Retail sampling



2.2.1. Traditional approaches

The quality of food products can be determined by combining sensory analysis with

specific microbiological and chemical tests (Suvanich et al., 2000). However, sensory

profiles between the closely-related species are difficult to be characterized by a consumer

panel even if a trained. Recently, an integration of electronic tongues and noses is used to

describe the identification and classification based on flavor and aroma and other

measurements of quality (Deisingh et al., 2004; Gomez et al., 2007). The techniques rely

on the information obtained from the multi-sensor arrays as principal components analysis

and artificial neural networks.

The chemical composition and nutritive value such as moisture, crude protein, ash,

and fat can significantly distinguish the different species of certain fish and seafood (Hong,

1991; Celik et al., 2004). A study conducted on crabs by Tureli et al. (2000) for example,

revealed that the ratio of crude protein, dry matter, and crude ash in the meat of male crabs

was higher in swimmer crabs (Portunus pelagicus) than in blue crabs (Callinectes

sapidus). A different study revealed that significant differences existed in the protein

contents of claw and body meat between those two species (Gokoolu & Yerlikaya, 2003).

Since fatty acid composition is practically a reflection of the diet, its profile can be used to

discriminate wild from cultivated fish (Martinez et al., 2003; Moretti et al., 2003).

Nonetheless, composition and nutritive value may differ depending on region, habitat, sex,

age, stages of life or sexual cycles, seasonal factors, and diet (Tsai et al., 1984; Reddy et

al., 1991).

2.2.2. Protein based methods (Proteomics)

Proteomics emerged in the beginning of the 1990s due to the need for new protein

analysis methods. The proteome is defined as the entire protein complement expressed by a

cell type of an organism (Wilkins et al., 1996). Proteome research focuses on the structural

and functional analysis of the proteome and the interaction of proteins. This includes the

isolation, identification and characterization of all proteins expressed by the organism’s

genome. Proteome analysis could lead the way to explain the function of an organism

dynamically rather than statically. This is important since the protein compositions and

concentrations change from cell type to cell type, even within sub-cellular compartments.

Moreover, they differ between various stages of development (Ferguson et al., 1995).



Proteins (enzymes, myoglobin, etc.) have been widely used as species markers.

Applicable techniques include separation of water-soluble proteins by starch,

polyacrylamide or agarose gel electrophoresis, IEF, and two-dimensional (2D)

electrophoresis (O’Farrell, 1975; Pineiro et al., 1999; Martinez et al., 2005 & 2007).

Highly resolved water-soluble protein patterns can be used to differentiate genetically

close-related species. Interestingly, the limit of detection of gel electrophoretical methods

varies between 0.1% and 1% (Xie, 2003). Notably, proteomics can be used to differentiate

species, breeds, and varieties by their specific protein pattern.

One and 2D protein electrophoresis are the most frequently used techniques for

proteome research, while peptide mass fingerprinting analysis by MALDI-TOF mass

spectrometry has become the most powerful techniques for proteome analysis (Boucherie

et al., 1995; Xie, 2003). Immunological techniques, like ELISA, performed on the solid

surface of microwell plates are using suitable target proteins for analysis. A qualitative

detection of animal species is possible and the limit of detection depends upon their

content in meat products, for instance in pork 61%; poultry and beef 62%; sheep 65%

(Schwagele, 2005).

Other protein separation and quantification techniques include liquid chromatography

(LC/HPLC) and capillary electrophoresis (López, 2007). Nevertheless, electrophoresis has

some drawback to identify processed products. Variation in factors, such as temperature,

pasteurization, and processing parameters, affect the results (Rehbein, 1990; Barlett et al.,

1993; Xie, 2003). In addition, immunological methods may generate cross-reaction among

protein from closely related species (Wolf et al., 2000; Necidová et al., 2002).

2.2.3. DNA based methods (Genomics)

The field of genomics utilizes a variety of technologies to study the information

content of cells. The genome research generally refers to sequencing the total genomic

DNA of an organism and mapping all genes within these sequences (Brooker, 1999). In

contrast to the proteome, genomic research focuses on the structural and functional

analysis of the gene as well as their recombination. This includes the isolation,

identification and characterization, either physical or genomic mapping, of the entire

genome (Johnson & Browman, 2007). Historically, the term “genome” was emerging in

1970s with the introduction of techniques for manipulating DNA (recombinant) and

reading its sequence (Watson & Berry, 2003).



DNA-based techniques have been demonstrated as fast, cheap and straightforward

gene identification (Mackie et al., 1999; Weder et al., 2001). Using DNA has many

advantages as it is unique to the individual and creates a means of permanent identification

throughout the life cycle of the animals. DNA traceability infrastructure could also be used

to check for the presence of a particular genetic modification (Loftus, 2005). In some

European markets, a routine program of verification sampling and DNA analysis/matching

is used to monitor the ability of a supply chain to provide traceable products. The

significant advantage of DNA-based methods is the applicability for identification of not

only fresh and frozen products, but also processed, degraded and mixtures, which are not

well achieved by protein-based method. Figure 2-2 reveals the link between different

identification techniques and their tools (Dupont et al., 2007).

Figure 2-2. Link between different identification techniques and their tools (Dupont et al., 2007) ESTs: expressed sequence tags

In industrial scale, DNA based traceability has been applied on fresh beef in the UK

and Ireland since the late 1990s (Hanluain, 2001). Recently, DNA barcoding has globally

gained much attention. Founded in 2004 at University of Guelph (Ontario), Barcode of

Life Data System (BOLD) establishes an integrated platform for species identification

based on DNA profile. The concept of DNA barcoding has been applied on North

American bird species (Hebert et al., 2004), Australian fish (Ward et al., 2005), and a

variety of other invertebrate (Barrett & Hebert, 2005; Smith et al., 2005). Development of

DNA barcoding systems will make large-scale applications of DNA traceability

increasingly cost effective and feasible.

Community

Species

Proteomics

Transcriptomics

Genomics

Metagenomics

mRNA

DNA

Protein

Toolbox

Sequencing Barcoding

ESTs Microarrays

1D & 2D Gel Electrophoresis



2.2.4. Other methods Visible and Near-Infrared (VIS/NIR) spectroscopy has been used to detect

adulteration in many high-value foods. VIS/NIR spectroscopy is an affordable, powerful,

and objective tool that requires minimal sample preparation and can creates fast results. In

addition, it does not require highly trained people to operate the instrument and interpret

the results. Few biological compounds absorb light in the visible region (380 – 700 nm),

which is usually function-related absorption (Pasquini, 2003). In contrast, biochemical

compounds such as DNA and RNA absorb light in the near ultraviolet range, though

absorption is unrelated to function. Absorption in the near-infrared region (780-2500 nm)

arises from vibration motion of hydrogen molecules (Gayo, 2006).

VIS/NIR technology has been used to determine the authenticity of olive oil that was

adulterated by other vegetable oils (Tay et al., 2002) and adulterated honey by sugar

solutions (Kelly et al., 2004). In fishery products, VIS/NIR spectroscopy has been used to

detect economic adulteration of Atlantic blue and blue swimmer crab meat which is

adulterated with surimi-based imitation crab meat (Gayo, 2006). Similarly, nuclear

magnetic resonance (NMR) can be used to obtain lipid fingerprints. By using this

technique, Martinez et al. (2003) revealed some nutraceutical fish oil capsules were

incorrectly labeled concerning the species and the lipid content.

The variations of isotopic abundances, such as hydrogen, carbon, nitrogen, and

oxygen, are of particular interest for food authentication studies. Since the variation of

isotopic abundance in animals is mainly due to the origin, nature of the feed, and animal’s

metabolism, it can be used both for origin assignment and to detect adulteration. The

isotope ratio of C and N has been used to examine the authentication in wild and farmed

Atlantic salmon (Dempson & Power, 2004) and European sea bass/Dicentrarchus labrax

(Sweeting et al., 2007).

2.3. Genomic identification based on mitochondrial DNA

Mitochondria are essential organelles in the cytoplasm since they serve many

important functions for the cell especially oxidative ATP-production, degradation of fatty

acids, and modulation of intracellular calcium homeostasis. They also play a major role in

cell signaling and apoptosis, as well as in biosynthesis (e.g. heme-groups, nucleotides, and

amino acids) and degradation (e.g. urea cycle) of metabolites (Kleinsmith & Kish, 1995).

ATP production is regulated in the citric acid cycle, also known as Krebs cycle. This is the

final common pathway for different metabolites such as carbohydrates, fatty acids and



amino acids. The compounds with a high redox-potential (reduced nicotinamide-adenine-

dinucleotide/NADH) and reduced flavin-adenine-dinucleotide (FADH2) are generated in

this cycle. The products are later delivered to the respiratory chain of the mitochondrion in

order to generate ATP.

Mitochondria are comprised of two membrane systems: intra (inner) and extra (outer)

membranes. In the center of the mitochondrion and between the membranes there are two

aqueous compartments: the matrix and the inter-membrane space (Kleinsmith & Kish,

1995). The two membrane systems contain carrier proteins and channels that regulate the

exchange of substrates between the compartments. The inner membrane is typically rich in

proteins in which the high molecular weight multi-protein-complexes of the respiratory

chain are located at the inner mitochondrial membrane. The total number of different

proteins or polypeptides making up a mitochondrion is estimated to be around 1000

(Kleinsmith & Kish, 1995).

Basically, a genetic profile or molecular identity can be provided from either nuclear

(nDNA) or mitochondrial genome (mtDNA). MtDNA has been successfully used as a

biological marker (Avise et al., 1987; Esposti et al., 1993; Lin et al., 2005) and has many

advantages:

1. MtDNA is unique as it is maternally inherited in most species. This means that only the

mtDNA of the oocyte is transmitted to the offspring. Exceptions with paternal leakage

exist in mice and double uniparental inheritance in sea mussels (Mytilidae), freshwater

mussel (Unionidae) and clam (Veneridae) (Hoeh et al., 1991; Burzynski et al., 2006).

2. MtDNA has a high mutation rate since it is vulnerable due to its compact structure,

lack of histone protection, insufficient repair mechanisms and exposure to reactive

oxygen species generated along the respiratory chain. This vulnerability results in the

higher mutation rate than the nuclear DNA (Zeviani et al., 1998).

3. The high copy number of mtDNA facilitates a successful analysis of degraded or very

limited amount of raw material (Watson & Berry, 2003).

4. Integrated genomic database of mtDNA has been established by various institutions,

such as NCBI, BOLD, CBOL’s, FISH-BOL, and Fishtrace.

MtDNA is a small circular molecule (Figure 2-3), generally between 14 and 18 kb

with exception in certain individual, such as sea scallop (family Pectenidae) which

contains up to +35 kb (Snyder et al., 1987). Typically, mtDNA is composed of 37 genes

coding for 22 tRNAs, 2 rRNAs (12S and 16S) and 13 mRNAs coding for proteins. The



mitochondrial genome is arranged very efficiently as it lacks introns and has small

intergenic spacers where the reading frames sometimes overlap. The control region is the

primary non-coding region, and is responsible for the regulation of heavy (H) and light (L)

strand transcription and of H-strand replication (Kleinsmith & Kish, 1995).

Figure 2-3. Illustration of mitochondria in a cell (left) and its genomic map (right)

Table 2-1. Applicable categorical levels of each molecular marker or gene region Kingdom Phylum Class Order Family Genus Species

A. Nuclear DNA 1. SSU (16-18S) +++++++++++++++++++++++++++------ 2. LSU (23-28S) ++++++++++++++++++------- 3. 5.8S +++++++++++++++++++++------ 4. IGS +++++5. ITS +++++++++++6. Rhodopsin*) B. Mitochondrial DNA 1. Ribosomal RNA - 12SrRNA +++++++++++++++++++------ - 16SrRNA +++++++++++++-----2. Protein 3. Coding genes - ND1 -------++++++++++++++++++ - ND2 -------++++++++++++++++++ - COI**) -------++++++++++++++++++ - COII -------++++++++++++++++++ - Cyt b*) -------++++++++++++++++++4. Control region +++++

The bold lines indicate as mostly applicable categorical levels for each molecular marker or gene region, while the dot lines indicate less frequently applicable *) : FishTrace; **) : Barcodinglife

Mitochondria Nucleus

Control region (D-Loop)

16SrRNA

12SrRNA Cyt b

ND6

ND5

ND4

ND4L

ND1

COII ATPase6

COIII ND3

22 tRNA genes

COI

12 protein-coding regions

ATPase8

ND2



Analysis of mtDNA is commonly accomplished by sequencing or fingerprinting

techniques of particular coding regions. Ribosomal RNA (12SrRNA and 16SrRNA),

cytochrome oxidase, cytochrome b, and D-loop or control region are commonly employed

for species identification (Passarino et al., 2002; Scander & Halanych, 2003; Watanabe et

al., 2004; Barlaan et al., 2005; Trotta et al., 2005; Goldenberg et al., 2007). Table 2-1

describes the applicable categorical levels of each molecular marker or gene region for

species identification (Hwang & Kim, 1999; FishTrace; Barcodinglife). In recent time,

mtDNA has been widely used for species identification of fish and seafood products which

are mostly focused on COI and cyt b regions (Ward et al., 2005; Ratnasingham & Hebert,

2007; Roe & Sperling 2007; Sevilla et al., 2007).

2.4. Fish and seafood authenticity based on the cytochrome b gene

Cytochrome b is one of the cytochromes involved in the electron transport in

respiratory chain of mitochondria. Located between tRNA-Glu and tRNA-Thr, cyt b

contains eight transmembrane helices connected by intra membrane or extra membrane

domains. The combination of cyt b gene and other genes in genomic DNA encodes for

cytochrome c oxidoreductase, which is a complex enzyme in oxidative phosphorylation

(Kleinsmith & Kish, 1995). Cyt b gene as a phylogenetic probe is widely used because it is

easier to align a protein-coding sequence that has evolved over the period than to align

either mitochondrial rDNA or noncoding sequences (Irwin et al., 1991). Kocher et al.

(1989) have shown that some highly conserved regions on the mitochondrial cyt b gene are

suitable for species identification in most vertebrate species.

The wide use in phylogenetic study has resulted cyt b as a universal marker. So far,

cyt b has been the most prevalent source of sequence data in fish (Bartlett & Davidson,

1991; DeSalle & Birstein, 1996; Rehbein et al., 1997; Céspedes et al., 1998; Hsieh et al.,

2007; Sevilla et al., 2007). However, the successful application of genomic based

traceability heavily depends on DNA sampling and DNA analysis. Generally, DNA based

identification mainly relies on the application of PCR to generate desired fragment of

specific region of DNA template.

2.4.1. Specimen and treatment of sample

DNA isolation is one of the important parts in PCR application since it is used as

template for PCR amplification. The DNA sampling method should provide high-quality



and high-quantity DNA useful to accommodate the targeted region. Basically, DNA isolate

can be obtained from any biological material. Technically, DNA sampling must be low-

cost, relatively easy to perform and produce samples in a format suitable for laboratory

analysis (Loftus & Laronde, 2005). However, non-destructive DNA isolation is desirable,

especially when working with live samples, or with threatened or endangered species,

where sacrificing the animals is unaffordable (Wasko et al., 2003).

There have been a number of innovations in DNA sampling specimen conservation.

Muscle tissue (mostly white muscle) represents the most common used sources of fish

DNA. Meanwhile, egg, liver and blood are used when the individuals are too small to be

effectively sampled by muscle (Barlett & Davidson, 1992; Martinez et al., 1998; Watanabe

et al., 2004). Moreover, sampling strategy for non-destructive DNA isolation can be

achieved from fish fins or scales (Wasko et al., 2003). Lysis buffer may be used for long

periods of delay before the specimen is extracted. The most used lysis buffer for specimen

preservation contains Tris-HCl, NaCl, EDTA, and SDS (TNES) either with or without urea

(4 to 8 M). In addition, formaldehyde has been recognized for fixing and preserving

biological specimens, especially for museum collection. These preserving materials have

been used to store fin and scale samples of fish for years with good DNA quality (Asahida

et al., 1996; Schander & Halanych, 2003).

2.4.2. Isolation of DNA

The isolation of highly-quality DNA is the major step for various molecular-biology

techniques. Low amount of DNA with the presence of PCR inhibitors are common factors

to compromise the PCR amplification (Weder, 2002; Di Pinto et al., 2007). Proteins,

RNAs, lipids, polysaccharides, and other leftover cellular constituents, are frequently

present in the DNA, particularly when isolated by classical methods. During PCR

amplification, these contaminants may interfere with restriction enzymes, ligases, and

thermostable DNA polymerase (Merente et al., 1998).

Rationally, DNA isolation procedures should aim to solubilize cellular components

and simultaneously inactivate any intracellular nucleases to conserve biologically active

DNA. Most isolation procedures combine the use of one or more agents, such as organic

solvents, detergents, N-lauryl sarcocyl, chaotropic salts, urea, etc. Occasionally, β-

mercaptoethanol is added for protein denaturation (Merente et al., 1998). Recently,



numerous commercial kits are available for isolation of total DNA. The use of commercial

kit permits production of higher quality DNA isolate.

2.4.2.1. Tissue digestion

In general, DNA isolation employs a buffer containing one or more detergents, for

instance SDS, NP-40 or Triton X-100. These detergents are used to break down the cell

wall lipoprotein. Its anion-binding property permits detergent to precipitate some

negatively charged compounds such as proteins and polysaccharides. TNES is the most

used buffer for cell digestion. Several studies used 500-600 μl of TNES consisting of 10-

200 mM Tris HCl pH 7.5-8.0, 125mM NaCl, 10-100mM EDTA, and 0.5-1% SDS to

extract 50-100 mg of fish tissue, fin and scale (DeSalle et al., 1993; Asahida et al., 1996;

Hsieh et al., 2003). SDS as detergent breaks apart fatty membranes of the cells while NaCl

(source of salt and metal ion) is intended to increase the osmotic pressure outside the cell

and help break apart the membranes. Concentrated salt solution changes the polarity of the

solution where DNA dissolves in, whereas fats, carbohydrates and proteins do not.

Since DNase is dependent on Mg2+ and Ca2+, the presence of EDTA (at least 2mM)

in lysis buffer is critical to chelate these cations and thereby prevents the degradation of

high molecular weight DNA (Merente et al., 1998). In addition, buffers maintain the

solution in alcaline pH to retain DNA in aqueous phase. The presence of SDS, EDTA,

metal ions and certain pH range is critical for the activity of enzymes involved in the DNA

extraction (Deshpande et al., 2001). Occasionally, 4-8 M urea is involved to breakdown

hard tissues such as fins and scales (Asahida et al., 1996; Hsieh et al., 2003).

Alternatively, guanidinium thiocyanate is often used for DNA extraction as it has

strong chaotropic properties (Botwell, 1987). Introduced by Chomczynki and Sacchi

(1987), it is used as protein denaturant, acting as RNase inhibitor, and protecting cellular

transcripts from degradation during tissue extraction. Nevertheless, since guanidinium

thiocyanate is categorized as harmful chemical, working with this method needs extra

protective equipment, such as goggles, fume hood, and proper gloves.

CTAB (cetyltrimethyl ammonium bromide) is frequently applied as surfactant in

DNA extraction. It is used for tissue digestion, together with β-mercaptoethanol and

polyvinylpyrolidone (PVP), followed by phenol-chloroform extraction. Originally, this

method is developed for extraction of polysaccharide-contaminated samples, essentially

plant tissue (Doyle & Doyle, 1987; Tel-Zur et al., 1999). PVP or activated charcoal or a



combination of both is often used in order to remove polyphenolics from further extraction

steps. This method is, besides laborious, limited by the use of hazardous reagents.

Konat et al. (1990) described a method that embeds the nuclei in agarose in order to

protect the DNA from mechanical shearing during isolation. In this procedure, Proteinase

K in the presence of SDS is used to remove proteins and lipid from the embedded DNA.

The limitation of this method is that it is only suitable for suspension cells, neither for

adherent cells nor tissues. The requirement of numerous buffer changes also makes this

method laborious and time consuming (Merente et al., 1998).

2.4.2.2. Separating proteins and contaminants

Proteinase K and RNase are two important enzymes involved in DNA extraction.

Proteinase K is essential to remove protein from the embedded DNA while RNase is

important to disrupt RNA that could interfere in the downstream applications. Proteinase K

is an endolytic protease that cleaves peptide bonds at the carboxylic sides of aliphatic,

aromatic or hydrophobic amino acids. Proteinase K is active in the presence of metal ions,

SDS, urea, chelating agents (e.g. EDTA), sulfhydryl reagents and trypsin or chymotrypsin

inhibitors. In spite of stable over a wide pH range (4-12.5), Proteinase K autolysis occurs

increasingly at alkaline pH. The optimum temperature for proteinase K is 55-65°C

(Merante et al., 1998). In contrast, most other enzymes (e.g. DNase, RNase) are denatured

under this condition. Therefore, the combination of Proteinase K and detergent buffer in

elevated temperature are essential for the inactivation of endogenous nuclease.

Commercially, proteinase K is produced by fungus Tritirachium album (Ebeling et al.,

1974).

Ribonuclease (RNase) is an endonuclease that cleaves the 3’ end of cytosine (C) and

uracil (U) residues. It is commonly used in molecular biology applications such as the

removal of contaminating RNA from DNA preparations (Lee et al., 1996). RNases are

ubiquitous in the environment as they are present in many biological materials. For

example, the pancreas is rich in RNase (>1 mg/g tissue) and is the source for most

commercially produced RNase. A study reported that RNase increases its activity in the

presence of approximately 2 M urea (Deshpande et al., 2001). Since the optimum

temperature of RNase is 37-45ºC, it must be applied separately from Proteinase K.

Likewise, Proteinase K rapidly inactivates nucleases including RNase. Once cellular



proteins and lipids are separated, contaminating RNA can be degraded by DNase-free

RNase followed by a chloroform extraction to remove the RNase.

Studies revealed that concentration of Proteinase K used for DNA extraction varies

depending on the kind of sample, time and temperature of incubation, and the presence of

RNase. A concentration of 50ng proteinase K per μl lysis solution was used to extract 50-

100mg of total DNA from fish tissue and fish fin with no addition of RNase, carried out at

37-42ºC incubation for overnight (8-16 hours) (Asahida et al., 1996). Wasko et al. (2003)

examined 75ng/μl proteinase for 100-300mg of fish fin and scales at 42ºC for 10 hours

followed by the same concentration of RNase (42ºC for 1 hour) while Hsieh et al. (2003)

worked with 400ng/μl on fish tissue without RNase. In addition, Imai et al. (2004)

employed relatively high concentration of Proteinase (4000ng/μl) for digesting crab (genus

Scyla) samples at 55ºC for 3 hours followed by 1000ng/μl RNase at 37ºC for 30 minutes.

2.4.2.3. Precipitation and recovery of DNA

In many classical DNA extraction procedures, organic solvents, mainly phenol and

chloroform, are used to remove contaminants. For this purpose, phenol must be used in

alkalic conditions (pH ≥ 7.5) otherwise the isolated DNA will remain in organic phase

and interface (Blin & Stafford, 1976). In practice, a phenol: chloroform mixture,

commonly added by isoamyl alcohol (P:C:I=25:24:1), at equal volume as lysis buffer are

added to DNA extract to form a biphasic mixture (Asahida et al., 1996; Hsieh et al., 2003;

Wasko et al., 2003). Isoamyl alcohol is important to prevent the mixture from foaming.

Through a centrifugation at high speed (>10.000 rpm for 5-10 minutes), proteins and lipids

will separate into the organic phase while the DNA, and other contaminants such as salts

and sugars, remain in the aqueous phase. This is sometimes repeated depending on the

requirements of the downstream processes, and then followed by extraction with

chloroform: isoamyl alcohol (C:I=24:1) to remove the residual phenol.

The aqueous phase containing DNA is concentrated by double volume of absolute

ethanol to precipitate the DNA. The polarity of DNA renders insoluble in ethanol which is

relatively less polar. The DNA precipitation is due to the interaction between ethanol and

water resulting in less capability to dissolve the DNA. Isopropanol can be used instead of

ethanol. In spite of the higher precipitation efficiency than ethanol, isopropanol is less

volatile and therefore needs more time to dry the isolated DNA. By centrifugation, the

DNA is concentrated and forms a white or transparent pellet. The next step is purification



using 70%-80% ethanol to remove salts present in the pellet. After air dried, purified DNA

is re-suspended in TE (Tris-HCl and EDTA) buffer.

Phenol-chloroform based extraction is proven to produce high yield of DNA with

relatively good quality for PCR and downstream applications (Asahida et al., 1996; Wasko

et al 2003). However, beside relatively laborious due to numerous changes of phenol and

chloroform, this precipitation method has drawback as phenol and chloroform are

hazardous and inconvenient materials. Thereby, extra caution is critical in order to handle

the problems.

2.4.2.4. Commercial kits

Many companies have developed commercial kits for total DNA extraction and

purification. The protocols that are available commercially for example Wizard DNA

Extraction kit and Wizard Magnetic DNA Purification for Food (Promega; Milan, Italy),

DNeasy Tissue kit and QIAamp DNA mini kit (Qiagen; Hilden, Germany), Chelex (BioRad;

Marne la Coquette, France), ABI PRISM 6100 Nucleic Acid Prep Station (Applied

Biosystems Inc.), and genomic DNA purification kit (BioNobile; Turku, Finland).

Based on the type, commercial kits can be classified in three categories i.e. manual

commercial kit, manual automated, and fully automated (Mattocks, 2007). Factors

governing the choice of kit are the speed of extraction, cost, reliability, sample

requirement, sample tracking, storage requirement, and the quality of isolated DNA. Most

of manual commercial kits work based on liquid-liquid phase extraction. The difference

with classical procedures is the use of ready-to-use reagents (lysis buffer, precipitation

reagents/washing buffer, and elution buffer) which minimize the work and time of

preparation. Moreover, commercial kits allow convenient work as they commonly offer

simpler procedures to provide purer DNA with safer reagents, importantly in avoiding the

use of phenol-chloroform.

In comparison to manual commercial kits, manually automated one has the

advantages of the use of liquid-solid phase, instead of liquid-liquid phase extraction. Silica

based column and magnetic beads are two important solid phases used to embed and bind

the DNA. Column based methods generally rely on the use of lysis solution, binding

buffer, washing buffer and elution buffer. After digestion with lysis solution, guanidinium

thiocyanate is commonly used as chaotropic salt to bind the DNA. Bound DNA is purified

by washing with buffer containing ethanol (Bio-Nobile, 2007). A special silica-glass



column then binds the DNA prior to its elution using a low salt buffer. This protocol

eliminates the need of organic solvent extraction, DNA precipitation, and minimizes

centrifugation (Roche, 2003).

The DNA-bead interaction is based upon the specific affinity of the ligand on the

surface of the beads. The beads can be coated with binding elements such as primary or

secondary biotinylated antibody, streptavidin and protein (A or G). Streptavidin beads

attach via a DNA linker simplifying bead removal by cleavage with DNase (BioNobile,

2007). Streptavidin magnetic beads present in different sizes (50 nm-3 µm in diameter

depending on the type of sample: cells, nucleic acids, or proteins) allow to minimize non-

specific binding of nucleic acid or protein to help prevent bead aggregation. Since the

separation of the beads from the aqueous phase occurs with a magnet, centrifugation is not

required. These kits may obtain DNA in less than one hour.

It has been shown that DNA analysis by a fully automated extraction, BioRobot EZ1

DNA Tissue kit (Qiagen Corporation, Valencia, CA) and MultiProbe II Plus EX (Perkin

Elmer, USA) for instance, proves time saving and reduces the risk of possible

contamination (Pizzamiglio et al., 2006). The automation of the process allows minimizing

manual interactions by means of robotic liquid handling, integrated shaking, heating, and

vacuum filtration.

2.4.3. Determination of DNA yield and purity

Several methods have been established for determination of DNA quantity and

quality, such as spectrophotometric methods, radioactive labeling, and fluorimetric

methods. Traditionally, spectrophotometry (UV-Vis) and spectrofluorometry are

techniques commonly used for quantifying DNA due to their simplicity and less cost.

Spectrophotometry technique measures DNA based on the determination of absorbance at

260 nm, the maximum absorbance of nitrogenous bases under UV light (Figure 2-4). On

the other hand, spectrofluorometry relies on the fluorescent signal created by fluorescent

dye-dsDNA binding at 480nm (excitation) and 520nm (emission). A number of reagents

are used as fluorescent dye including ethidium bromide (EtBr), cyanine, Hoechst-33258,

protamine, and hypocrellin A (Zhu et al., 1997).

Both spectrophotometry and spectrofluorometry have advantages and disadvantages.

It has been shown that spectrophotometry is the cheapest way to quantify DNA

concentration and also simple as it can be acquired without the use of reference/DNA



standard. Since the concentration of pure dsDNA is 50μg/ml (when A260=1.0),

concentration of unknown DNA can be formulated as follows (Merante et al., 1998):

DNA (μg/ml) = A260 x l (light path in cm) x dilution factor x 50

The major disadvantages of the spectrophotometric method are the contribution of

nucleotides and single-stranded DNA to the signal, and the inability to distinguish between

DNA and RNA. Conversely, fluorometric method has higher sensitivity since it only

measures dsDNA embedded by fluorescent dye. Generally, the detection limit by classical

spectrophotometer is around 5 μg/ml dsDNA aliquot, while by spectrofluorometer it is

10ng/ml (Paul & Myers, 1982; Zhu et al., 1997). Certain dyes even demonstrate the

detection limit as low as 25pg/ml (Invitrogen, 2007). Fluorometric method is suitable for

selective targets, dsDNA, ssDNA or RNA, with less interference. Nevertheless, the use of

this method is restricted by the relatively high cost of fluorescent reagents.

A UV-vis spectrophotometer allows measuring DNA quality/purity based on the ratio

of absorbance at 260 and 280nm (A260/280). Theoretically, contaminating protein absorbs

UV light at +280nm whereas phenol at +270nm. Contaminated DNA with any of these

molecules expresses increasing absorbance at 280nm. The A260/280 ratio between 1.8 and

2.0 suggests minimal contamination (Smith, 1998). Lower values indicate protein

contamination, higher values RNA contamination.

Figure 2-4. Characteristic spectrum of DNA obtained by spectrophotometer (left) and spectrofluorometer (right)

Gel electrophoresis can also be used to evaluate DNA purity as well as to estimate

DNA concentration. By loading onto a 0.8-1.5% agarose gel, DNA fragments can be

separated by electrophoretic charge based on their lengths (molecular weight). The

intensity of the bands correlates to the DNA concentration. More recently, there has been

Wavelength (nm) Wavelength (nm)

260 nm

dsDNA

OD

ssDNA RNA

Ex:480nmDNA

500 600 700200 225 275 250 300 320

0.3

0.6

0.9

1.2

1.5



increasing interest in the use of alternative techniques of DNA quantification such as

quantitative PCR, hybridization, pulse-field gel electrophoresis (PFGE), and threshold

assay. These techniques are proven to be useful due to a lower detection limit and their

robustness (Mehta & Keer, 2007).

2.4.4. Polymerase chain reaction

The study of product authenticity based on genomic profiling has been rapidly

developed and improved with the progress of molecular biological tools, in particular

polymerase chain reaction (PCR). Invented in 1983 by Kary Mullis at the Cetus

Corporation, it offers a simple process to massively enrich a targeted fragment of DNA

(Watson & Berry, 2003). PCR is an in vitro DNA amplification that involves a repeated

cycling process of defined stages, termed denaturation, annealing, and extension. The

reagents required for the PCR include DNA polymerase, each of four nucleotides (dNTPs),

a primer couple, magnesium source, buffer solution and DNA template (Rapley, 1998).

Rationally, when DNA is heated in excess of 94-95ºC for at least 60 seconds, the

double strands come apart to produce two single strands (Rapley, 1998; Watson & Berry,

2003). This process is called denaturation, which allows the primers to bind to the DNA

template (annealing), whose sequences are complementary to the primers. In the next stage

the temperature is reduced to 35-60ºC for 30-120 seconds. Subsequently, the polymerase

makes a complementary copy of the template DNA started from each primer, thereby

creating a double strand of the target region. Known as extension, this step usually takes

place at 72ºC for 60-180 seconds. In the next cycle, the dsDNA produced from the

previous cycle becomes new template to produce a double new dsDNA (Figure 2-5).

Figure 2-5. The principle of PCR amplification

1st Cycle 2nd Cycle, etc...

primer

DNA template Add DNA polymerase

Add DNA polymerase

Separate 2 DNA strands, add primer

Separate 2 DNA strands, add primer

primer

primer



Recently, real time PCR (RT-PCR) has been widely applied for DNA amplification

due to its robustness, simplicity, and sensitivity (Weder et al., 2001; Hsieh et al., 2007).

RT-PCR allows both qualitative and quantitative analysis. This technique incorporates the

amplification and fluorescent detection by a single instrument in a single tube with data

recorded online. Hence, the use of gel electrophoresis to assess the targeted product can be

eliminated.

Real-time and traditional PCR methods have been developed into a key technology

for identification of foods, including fish and seafood products. Specific DNA sequences

of fish and seafood have been generated through PCR amplification by designing site-

specific primers, such as tuna/family Scombroidae (Bartlett & Davidson, 1991; Rehbein et

al., 1997; Richardson et al., 2006), caviar (DeSalle & Birstein, 1996; Rehbein et al., 1997),

salmonoid (Rehbein et al., 1997), flatfish (Céspedes et al., 1998), mollusc and arthropods

(Meritt et al., 1998), puffer fish (Cheng et al., 2003; Hsieh et al., 2003), gadoid/family

Gadidae (Aranishi et al., 2005), billfish/family Istiophoridae (Hsieh et al., 2005;

Richardson et al., 2006) and teleost fish (Sevilla et al., 2007).

2.4.4.1. Primer design

Since annealing is started from the primer, it becomes a critical part in PCR

amplification. Primers are designed to recognize the targeted region. The primers used in

the PCR are designed based on existing sequences of close similarities or evolutionary

conserved sequences. Many online sequences can be obtained from genetic databases, such

as GenBank, EMBL, BOLD, MitoFish, and CBOL’s. Specifically, sequences of fish and

seafood are available via FISH-BOL and FishTrace. Amino acid sequence information

may also be used to design PCR primers (Rapley, 1998). Besides from GenBank, amino

acid sequences can be provided from Mascot, PeptideMass, ExPASy, and so on.

There are some considerations in designing primers. Generally, the primers should

have a matched “GC” content of 40-60% and must not have the potential to form primer-

dimers or hairpin beacons (Rapley, 1998). Table 2-2 describes a number of primers used

for the amplification of the cyt b region in fish and seafood products. Typically, 15-30 base

primers anneal efficiently if the PCR enzyme is Taq Polymerase, however this number

may change for enzymes with greater heat stability (Roche, 2006). Shorter primers (less

than 15 bases) anneal very effectively but they may not be specific enough. In contrast,

longer primers recognize targets specifically but tend to anneal with lower efficiency.

Priming efficiency can be increased by a G or C, or CG or GC at the 3' ends. Design of 3



or more C or G at the (3') ends should be avoided since it may promote mispriming at G or

C-rich sequences due to stability of annealing (Qiagen, 2005). Primer pairs are designed

with matched melting temperature (Tm). The difference of 5oC or more leads to reduce the

amplification (Rychlik et al., 1990). Tm and optimum annealing temperature (Ta,Opt) can be

estimated as follows:

Tm (ºC) = 2 x (number of [A+T]) + 4 x (number of [G+C])

Ta,Opt (ºC) = 0.3 x(Tm of primer) + 0.7 x(Tm of product) – 25

Table 2-2. Various primer couples used to amplify cyt b region of fish and seafood

Name of primer Sequence (5’-3’) Size

(mers)

Targeted Fragment

(bp)

Target species and Source of reference

L14841 H15149

AAAAAGCTTCCATCCAACCAACATCTCAGCATGATGAAAAAACTGCAGCCCCTCAGAATGATATTTGTCCTCA

31 30 376 Vertebrate species

(Kocher et al., 1989)

Cyt BL Cyt BH

CCATCCAACATCTCAGCATGATGAAA CCCCTCAGAATGATATTTGTCCTCA

26 25 358 Tuna/family Scombroidae

(Bartlett & Davidson, 1991)

Cyt BL1 Cyt BH

CCATCCAACATCTCAGCATGATGAAA CCCCTCAAAATGATATTTGTCCTCA

26 25 358 Salmon, mackerel, hering, cod

(Bartlett & Davidson, 1992)

FB349 FB496

GTCGAATGAATCTGAGGAGGCTT CCRATTGGGTTGTTTGACCCTGTTTC

23 26 148 Tuna, bonito, mackerel

(Rehbein et al., 1995)

59-3 59-4

AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA GCTGGTACCTCTACAAAGAAACATGAAACA

34 30 123 Cod, herring, sardine, tuna,

bonito (Rehbein et al., 1997)

CytB1 CytB2

CCATCCAACATCTCAGCATGATGAAA CCCCTCAGAATGATATTTGTCCTCA

26 25 358 Flatfish

(Céspedes et al., 1998)

UCYTB144F UCYTB272R UCYTB151F UCYTB270R

TGAGSNCARATGTCNTWYTG GCRAANAGRAARTACCAYTC TGTGGRGCNACYGTWATYACTAA AANAGGAARTAYCAYTCNGGYTG

20 20 23 23

430 Mollusc and arthropods (Meritt et al., 1998)

TR-14F TR-571R

GGAAAACCCATCCAATCCTA CAGCAACAACAAAGGGGAAT

20 20 558 Gadoid/family Gadidae

(Aranishi et al., 2005)

L-CYTBF H-CYTBF

GCTATRCACTAYACMTCRGAC GCCTCCTCARATTCATTGGAC

21 21 348 Billfish/family Istiophoridae

(Hsieh et al., 2005)

CBF-A CB3-3

CCCTCTAATATCTCQGTCTGATGAAACB GCGTAGGCAAATAGGAARTATCAYTC

28 26 715 Scombroidae, Istiophoridae

(Richardson et al., 2006) CytBI-6F CytBI-7F CytBI-1F CytBI-5R CytBI-3R CytBI-2R CytBI-4R

TTCTCAGTAGACAACGCCACCCT CTAACCCGATTCTTTGCCTTCCACTTCCT CGATTCTTCGCATTCCACTTCCT GGTCTTTGTAGGAGAAGTATGGGTGGAA GGGGTAAAGTTGTCTGGGTCTCC GCGGGGGTAAAGTTGTCTGGGTC AGGAAGTATCATTCGGGCTTAATATG

23 29 23 28 23 23 26

700-750 Teleost fish (Sevilla et al., 2007)

Wobbles: Y: T/C; M: A/C; R: G/A; K: T/G; W: A/T; S: C/G; H: A/C/T; V: A/C/G; D: A/G/T; B: C/G/T; N: A/C/G/T



Not only for specific amplification, but primers are also important for post-PCR

application, such as direct sequencing and fingerprinting analysis. In some cases, a

degenerate primer is useful to overcome the limited sequence information of such

species/families (Knoth et al., 1988). By introducing alternative bases (wobbles) as inosine

at a particular position, degenerate primers are advantageous for amplification of wide

range of families (Crick, 1966). A number of computer software are currently available to

aid in primer design, for instance DNAsoftware, PrimerDesign, EMBL, PremierBiosoft,

and OligoDesign.

2.4.4.2. Components of PCR reaction

During PCR amplification an enzyme is used to carry out the extension of the

fragment. Historically, DNA polymerase-I was used for this purpose. However, because it

is heat labile, fresh enzyme was required during each cycle which made the technique

laborious and costly (Rapley, 1998). The introduction of thermostable DNA polymerase

transformed PCR technique to allow full automation. The first introduced thermostable

DNA polymerase was isolated from the bacterium Thermophylus aquaticus, namely Taq

DNA polymerase which has optimum temperature of 72ºC (Eckert & Kunkel, 1990). The

higher thermal stability, the more useful is the enzyme, especially for amplifying GC-rich

regions where a high denaturation temperature is required. Several thermostable DNA

polymerases are produced from the different sources, such as Thermococcus litoralis,

Thermotoga maritime, and Thermus thermophilus (Rapley, 1998).

Magnesium (Mg2+) is another important element in the PCR amplification. It is not

only required to form a complex with the dNTPs which is critical for incorporation in the

extension step of the PCR cycle but it also affects the specificity of the primer-template

interaction and the denaturation of the dsDNA (Rapley, 1998). Insufficient Mg2+ results in

low yields while an excess of it creates nonspecific products. Generally, a concentration of

1-4 mM MgCl2 is used, depending on the DNA template and the primers.

The pH of the PCR reaction is an essential factor since changes in pH will affect the

amplification efficiency, in particular of long fragments (Cheng et al., 1994). In order to

maintain the pH at 8.3, at room temperature, a buffer/salt containing 50 mM KCl and 10

mM Tris-HCl is used. The PCR efficiency may also be reduced by contaminants present in

the template or caused by improper handling.



Essentially, application of RT-PCR requires fluorescent dye to detect amplified

product. There are two classes of fluorescent assays in RT-PCR, known as sequence-

independent and sequence-specific probe. Sequence-independent assays rely on

fluorophores, SYBR Green I for instance, that binds to entire dsDNA molecule instead of

oligonucleotide. Conversely, sequence-specific assay rely on oligonucleotide probes that

hybridize to their complementary sequence in the targeted products. Fluorescent

Resonance Energy Transfer (FRET) and HyProbe are examples of sequence-specific

probes (Roche, 2006).

2.4.5. PCR product analysis

Traditional PCR requires post-run visualization to assess the targeted fragment

(amplified product). Generally, DNA strands are able to be separated and visualized by the

application of polyacrylamide-GE, agarose-GE, or pulse field-GE (Smith, 1998).

Confirmation of the targeted PCR product is normally performed by using GE. Agarose-

GE separates a wide range of DNA fragments (0.1-40 kb), which is preferable to

polyacrylamide-GE (5-400bp). An agarose concentration of 0.3-3% (w/v) in TAE buffer

1x is commonly used to separate DNA fragments. The longer the fragment the lower the

concentration of agarose required.

A set of apparatus is needed to carry out the electrophoresis. Recently, the submarine

gel system is universally used for agarose-GE (Smith, 1998). In this system, the prepared

agarose gel on a supporting plate is submerged into a chamber containing electrophoresis

buffer (TAE 1x). The wells are created in the agarose gel with the aid of a comb inserted

into the cooling agarose. Into these wells, samples which have been mixed with a loading

dye are loaded. Electrophoresis is carried out for certain period, varying considerably from

30 minutes to hours, depending on the size of the gel, gel concentration, and electrical

charge. The DNA fragment migrates from the negative charge to the positive charge.

An intercalating dye is included in the electrophoresis to visualize DNA fragments.

The dye intercalates the stacked bases of dsDNA to produce fluorescence when illuminated

under UV light (254-300 nm). A particular concentration of dye can be mixed within the

gel, running buffer or combination of both. EtBr (3,8-diamino-6-ethyl-5-phenyl-

phenanthridium bromide) was commonly used for DNA staining. However, EtBr is known

as powerful mutagenic and potential carcinogen. Recently, lower mutagenic dyes such as



SybrSafe, SybrGreen, SybrGold (Molecular Probes®) and GelRed, GelGreen (Biotium Inc.)

are established commercially, despite their costs are higher than EtBr.

A successful PCR amplification results a strong band of targeted amplified product

with faint bands of residual nucleotides and primers. Side bands or non amplifiable band

indicates that amplification is not optimal. Overload of DNA will result in smear-bands

while excessive salt will lead to necking bands (Smith, 1998). Since GE only assesses

targeted fragment, it does not give species/product specific information. Further analysis is

required to assess the amplified products, particularly based on the nucleotide profiles.

Direct sequencing and fingerprinting techniques are commonly used for analyzing PCR

product.

2.4.5.1. DNA sequencing

DNA sequencing is the most informative and precise technique for species

identification. DNA sequencing is a modification of DNA replication, which differs from

normal replication in the inclusion of dideoxynucleotides (ddNTPs) instead of dNTPs

(Sanger et al., 1977). The different between ddNTP and dNTP is the lack of a 3’-OH

group. When ddNTP is incorporated into DNA, the synthesis is terminated, the process

known as termination sequencing. Sequencing is carried out in four reactions, each of

which contains all four dNTPs but only one of ddNTP. Thus, the reaction products are four

nested sets of fragments which are terminated according to the ddNTP included in the

reaction.

Historically, sequencing involves cloning the DNA into a suitable vector to create

DNA in a format that can be used for sequencing. There are two categories of cloning

vectors, known as single-stranded and double-stranded vectors. Plasmid of a bacterium,

such as E. coli, is the most used double-stranded vector for DNA cloning. On the other

hand, a single-stranded vector can be obtained from viruses or bacteriophages (Rapley,

1998). Recently, PCR products can be used as a sequence template. This technique, called

direct sequencing, eliminates the cloning stage and thereby significantly saves time and

materials. The disadvantages of direct sequencing are the limited fragment size (1000-

2000bp) and the need of PCR products purification. Purity and sufficient concentration of

amplified product becomes the most critical factor for the efficiency and reliability of the

sequencing method (Rapley, 1998).



In order to visualize the sequence, a detectable label is incorporated into the

sequenced strands. There are three types of labels used to visualize the sequence:

radioactive, four-color fluorescent, and one-color fluorescent. Radioactive based labeling

uses isotope of 35S, 32P or 33P to be incorporated to the ddNTP. The extended DNA is

visualized after electrophoresis by drying the gel and subsequently exposed it into a film

sensitive to radioactivity. 35S is the most used isotope as it exposes low energy β-emission

with reasonable half-life (87.4d) (Spencer, 1998). Recently, the trend of sequencing is

moving toward automated-sequencing system based on fluorescennce. Fluorescent

sequencing relies on excitation of fluorescent-labeled DNA strands when exposed to laser

light. The major advantage of this technique is that the sequence information can be

collected directly while the gel is running (Spencer, 1998). Applied Biosystems/ABI

(Perkin Elmer Corp), Amersham Pharmacia Biotech, and Li-Cor Biosciences are among

recognized manufacturers of automated sequencer.

For species differentiation, the sequence of DNA template is compared to

references/libraries. The index of similarity between two or more sequences is used to

analyze the most likely identification by using BLAST (Basic Local Alignment Search

Tool). Sequencing enables species identification without reference material if the generated

sequence is available in the database. The database can be created manually or obtained

online. The most recognized online DNA databases are GenBank and EMBL, while

Fishtrace specifically constructs sequence databases of commercially important fish based

on cyt b and nuclear rhodopsin. Nonetheless, not all sequences of the species are available

in several online DNA databases.

Direct sequencing has been successfully used to identify tuna species (Bartlett &

Davidson, 1991), adulterated dried mullet (Hsieh et al., 2003), specimens of tuna and

billfish larvae (Richardson et al., 2006) and teleost specimen (Sevilla et al., 2007) based on

the cyt b gene. Sequencing was also employed to identify Australian fish species for DNA

barcoding purposes based on COI region (Ward et al., 2005).

2.4.5.2. Fingerprinting techniques

The main drawback for large-scale implementation of sequence-based identification

is cost requirement. Alternatively, several methods have been developed for species

identification. These techniques, known as fingerprinting, include Denaturing/

Temperature Gradient Gel Electrophoresis (DGGE/TGGE), Single Strand Conformation



Polymorphism (SSCP), Restriction Fragment Length Polymorphism (RFLP), Amplified

Fragment Length Polymorphism (AFLP), and Random Amplified Polymorphic DNA

(RAPD) (Rapley, 1998; Spencer, 1998; Brooker, 1999; Mackie et al., 1999; Hird et al.,

2005). By using these methods, the phylogenic information can be obtained more

economically and rapidly (Hwang & Kim, 1999).

DGGE employs the use of a polyacrylamide gel with a gradient of denaturant to

separate the targeted DNA based on its stability against the denaturant. DNA fragments

rich in “GC” will be more stable and remain double-stranded until reaching higher

denaturant concentrations (Rapley, 1998). A mixture of ureum-formamide is used as

denaturants in the DGGE technique. Denatured DNA molecules become effectively larger

and slow down or stop in the gel and thereafter DNA fragments of different sequences can

be separated. The system is very sensitive to detect a single mutation in several hundreds

of base pairs of DNA (Smith, 1998). Similarly, TGGE is generated based on the gradient

temperature (Rapley, 1998).

PCR-RFLP is the most widely used method for identifying different fish (Hsieh et

al., 2003). This technique relies on the use of restriction enzymes which cleave dsDNA at

defined base pairs to generate species specific DNA fragments. Differences of base pair

between species can be detected by choosing a restriction enzyme which cuts the DNA at

particular sites. Endonucleases such as AluI, EcoRI, HaeIII, HindIII, SmaI, etc, are

commonly used for restriction analysis of amplified PCR products (Rapley, 1998). The

EcoRI, an enzyme produced by E. coli RY13 for example, recognizes every site consisting

“GAATTC”. After digestion for several hours at 37°C, the resulting fragments are

separated by electrophoresis in an agarose gel containing fluorescent dye. The advantage of

this method is that it is suitable for some functional genes which are not resolved by

DGGE analyses (Brooker, 1999). RFLP may also overcome problems with degenerate

primers that can plague some DGGE methods. PCR-RFLP enables to identify a single

point mutation since a restriction site would change the restriction pattern and a clear

identification becomes possible (Brooker, 1999). PCR-AFLP/RFLP analysis of the cyt b

gene has been used for identification of fish species (Ram et al., 1996; Cespedes et al.,

1998; Hsieh et al., 2003).

In SSCP, dsDNA is denatured by denaturing solution containing formamide,

bromphenol blue, and xylene cyanol in NaOH to form ssDNA. The ssDNA is then folded

such that complementary sections are bound together. Differences in the three dimensional



structure caused by alterations in base pair sequences of the single strand lead to differ the

electrophoretic mobility, which are visualized by silver staining. In comparison to other

fingerprinting techniques, SSCP is relatively cheap and fast to perform. Differentiation of

canned tuna and bonito has been achieved by this method (Rehbein et al., 1995). In

addition, the RAPD technique uses a short and not specific primer to generate unique band

patterns of amplified DNA (Mamuris et al., 1999). However, the use of SSCP and RAPD

techniques is limited by the necessity of reference material (Rehbein et al., 1995).

2.4.5.3. Other techniques

There are relatively new technologies which work in the similar way as finger

printing. Denaturing HPLC (PCR-DHPLC) employs an HPLC column rather than

acrylamide gel for separating PCR products (Barlaan et al., 2005. Separation of amplified

products is according to the elution of partially melted DNA bases. Since typically GC-rich

DNA denaturizes at higher temperature, it leads to the reduction of dsDNA and therefore

the elution of the sample will be faster. This technique was originally developed for

mutation analysis (Frueh & Noyer, 2003).

Not only it is able to generate targeted DNA fragment, RT-PCR also offers some

advantages in comparison to classical PCR. Online information, chiefly the amplification

curve and melting points, can be provided while PCR is running. Since melting point (Tm)

is basically sequence dependent, it permits to discriminate between different PCR

fragments. RT-PCR has been satisfactorily used to identify fish fillet from grouper, tuna

fishes, and commonly substitute species (Lopez & Pardo, 2005; Trotta et al., 2005).



CHAPTER III OBJECTIVES OF PRESENT STUDY

3.1. Problems

Primer design plays an important role in the PCR amplification and contributes

significantly to the downstream applications of product identification. The previous

literature reviewed various primers have been developed specifically to amplify specific

groups of fish and seafood products. However, several fish identities are not resolved by

existing primers, for instance Microstomus kitt, Scopthalmus maximus, Hyperoplus

lanceolatus and Ammodytes tobianus (Hoffman, personal communication). In addition,

studies on the genomic identification of elasmobranches, crustaceans and molluscs based

on cyt b gene are very limited. The studies on the identification of crustacean and

molluscan taxa focusing on cyt b were described by Meritt et al. (1998) and Nguyen et al.

(2002), while the studies on the identification of elasmobranches were focused on shark

(Martin & Palumbi, 1993; Heist & Gold 1999; Briscoe et al., 2005).

With regard to the fact that there is no universal primer couple available for the

majority of fish, crustaceans and molluscs at present time, it is essential to provide and

validate it. Universal primers are valuable for identification of samples that are

unrecognizable by morphological characters such as filleted products, surimi and other

processed/mixed products. It is also advantageous for identification of a wider range of

species or species that are not resolved by existing primers.

3.2. Objectives The main goal of this study is to develop standardized universal primers for species

identification of fish and seafood (teleost fish, elasmobranches, crustaceans and molluscs)

by polymerase chain reaction base on cyt b mitochondrial DNA. Furthermore, since DNA

quality, primers suitability and amplification condition are among the crucial factors in the

success of PCR amplification, this research aims to:

a. evaluate the different DNA extraction methods,

b. optimize the existing primers for cyt b gene amplification suitable for fish, mollusc and

crustaceans. Adapted primers were designed based on the evaluation of existing

primers, and

c. provide the optimal PCR protocol of the validated primers.



3.3. Research framework

: Focus of the study

Figure 3-1. Framework of present study

Sample collection

Morphological identification(if available)

Comparison of DNA extraction methods DNA isolation

PCR/RT-PCR amplification

PCR product analysis

RT-PCR analysis Sequencing

DNA quantity and purity

- Evaluation of existing primers

- Designing the degenerate primers

- Validation of universal primers

- Evaluation of targeted fragments - Index of similarity (BLAST)

- Evaluation of melting points (Tm) - Identification of dissimilarity

Sequence database

Unknown biological references



CHAPTER IV MATERIALS AND METHODS

4.1. Construction of reference database of cyt b gene

Genomic data bases of cyt b of various fish and seafood were collected via NCBI and

FishTrace (Figure 4-1). In NCBI, cyt-b database is categorized in ‘Nucleotide’ group on

menu ‘Search’. Species of to be searched fish/seafood must be entered, for example:

‘Gadus morhua cytochrome b’, then ‘Go’ must be clicked. The available database

(sequence) must be displayed in ‘FASTA’ to perform the sequences as fasta format. The

fasta format sequence is then copied to ‘Notepad’ in order to be able to integrate it into

bioinformatics applications. The format of entry files is ‘>gb nr|species name|reference

number|cytb|data origin (NCBI or FishTrace)’. Meanwhile, FishTrace’s data base can be

searched by either scientific name or common name. The cyt-b sequence is classified in

‘Genetics’ group. The detail sequence, furthermore, must also be copied as FASTA format

as explained previously. The provided sequences were used in designing the primers and

evaluating the sequence results.

Figure 4-1. Genomic database obtained from NCBI (left) and FishTrace (right)

4.2. Sample collection and preservation

Selected samples (finfish, molluscs and crustaceans) were obtained from either the

North Sea or selected fish markets in Belgium. Fresh samples were identified by means of

morphological features. Gutted and eviscerated samples were filleted prior to extraction.

Three to six specimens of 14 species of fish, 7 species of crustaceans, and 6 species of

molluscs were used for this work. Specification of the samples is described as follows:



a. Fish:

- Round fish: cod (Gadus morhua), faneca (Trisopterus luscus), tuna (Thunnus sp.),

and grey gurnard (Eutrigla gurnardus).

- Flat fish: common sole (Solea solea), lemon sole (Microstomus kitt), and tarbot

(Scophthalmus maximus).

- Elasmobranches: thornback ray (Raja clavata) and smooth skate (Malacoraja senta).

- Sand lances (smelt): small sandeel (Ammodytes tobianus), great sandeel (Hyperoplus

lanceolatus), and 3 unknown smelt fish (US1, US2, and US3).

b. Crustacean:

- Brown shrimp (Crangon crangon), giant tiger shrimp (Penaeus monodon), king

shrimp (P. Latisulcatus), giant river shrimp (Macrobranchium rosenbergii), deep-sea

shrimp (Solenocera sp), and 2 unknown shrimp (990 and 991).

c. Mollusc:

- Blue mussel (Mytilus edulis), green mussel (Perna canaliculus), illex squid (Illex

argentinus), and 3 unknown mussels (T1, 120 and 2251)

The reason for choosing these species was that some of them have previously been

well studied, such as G. morhua, Thunnus sp, and S. solea (Bartlett & Davidson, 1991;

Rehbein et al., 1997; Céspedes et al., 1998; Richardson et al., 2006). Other species are

known difficult to be identified by existing primers, for instance M. kitt, S. maximus, A.

tobianus, H. lanceolatus, C. crangon and M. edulis.

4.3. Comparison of DNA extraction methods

A commercial kit (Wizard DNA Extraction kit-Promega) and 3 classical methods

based on TNES-Urea Phenol-Chloroform were evaluated to isolate the total DNA of 4

selected samples (S. solea, T. luscus, C. crangon, and M. edulis). These classical methods

have been widely used to extract DNA either from fish tissue, fin, scales, or food products

(Asahida, et al., 1996, Wasko et al., 2003; Hsieh et al., 2005). The best method, resulting

in the best DNA quantity and purity for PCR application was then used in the further

application. The chemicals, reagents and solutions used for this work are described in

Appendix II (see page 70).



4.3.1. DNA extraction method 1 (Promega)

Isolation of DNA was performed based on the manufacturer’s instruction (Promega,

2002). 120 μl of a 0.5M EDTA solution pH 8.0 was added to 600 μl of Nuclei Lysis

Solution (NLS) in a 1.5 ml Eppendorf tube. Approximately 100 mg of tissue was added

into each tube followed by 17.5 μl of 20 mg/ml Proteinase K. The tubes were then

vortexed gently for 20 second and incubated overnight at 55ºC. Subsequently, 3 μl of 4

mg/ml RNase were added to the nuclear lysate and mixed gently prior to incubation at

37ºC for 30 minutes.

The DNA was extracted by adding 200 μl of Protein Precipitation Solution (PPS).

The tubes were then vortexed vigorously at high speed and followed by a centrifugation at

14.000 rpm for 10 minutes. Supernatant was transferred to a new tube containing 600 μl of

isopropanol (IPA). After being chilled at -20ºC for 1 hour, the DNA was precipitated at

14.000 rpm for 10 minutes. The DNA pellet was washed with 600 μl of 70% ethanol, air

dried and resuspended in 100 μl TE buffer.

4.3.2. DNA extraction method 2 (Hsieh et al., 2005)

In a 1.5 ml Eppendorf tube, approximately 100 mg of tissue was crushed with 600

μl TNES buffer (1% SDS). 15 μl of 20 mg/ml Proteinase K was added to each tube

followed by overnight incubation at 55ºC. The DNA isolation was performed by 600 μl of

phenol:chloroform:isoamyl alcohol (25:24:1). Rotation at 14.000 rpm for 10 minutes

separated the undissolved materials. The top layer was transferred to a new tube followed

by another extraction with 600 μl of phenol:chloroform (24:1). The DNA was precipitated

in 3M NaOAc pH 5.3 and two volumes of absolute ethanol. After being cooled at -20ºC for

1 hour, the tubes were centrifuged at 14.000 rpm for 10 minutes. The DNA pellet was

washed in 600 μl of 70% ethanol, air dried and resuspended in 100 μl of TE buffer.

4.3.3. DNA extraction method 3 (Wasko et al., 2003)

Approximately 100 mg of fresh tissue was grounded in a 15 ml falcon tube with 4 ml

of TNES-Urea buffer (0.5% SDS; 4 M urea). 15 μl of 20mg/ml RNase was added to the

tube prior to an hour of incubation at 42ºC. After incubation, 75 μl of 4 mg/ml Proteinase

K was added to each tube followed by incubation at 42ºC for 10 hours.



The DNA was extracted with 4 ml of phenol:chloroform:isoamyl alcohol (25:24:1).

After being inverted for 30 seconds, the tubes were rotated at 14.000 rpm for 10 minutes.

The top layer was transferred to a new tube. The DNA was precipitated in 1M NaCl and

two volumes of absolute ethanol. Centrifugation at 14.000 rpm for 10 minutes was done to

recover DNA. The DNA pellet was briefly washed in 70% ethanol, air dried and

resuspended in TE buffer and incubated at 65ºC for 1 hour.

4.3.4. DNA extraction method 4 (Asahida et al., 1996)

Approximately 100 mg of tissue was transferred into a 1.5 ml Eppendorf tube. 600 μl

of TNES-Urea buffer (6 M urea; 1% SDS) was used as the digestion buffer. 2μl of 20

mg/ml Proteinase K was added to the tube for protein digestion and the mixture then

incubated overnight at 37ºC. After this period, 3 μl of RNase was added to the tubes to and

the tubes were maintained at 37ºC for 30 minutes.

The DNA was isolated by adding 600 μl of phenol:chloroform:isoamyl alcohol

(25:24:1) to the tube. After being inverted for 20 seconds, it was rotated at 14.000 rpm for

10 minutes. The top aqueous layer was transferred to a new tube followed by the second

extraction by adding 600 μl chloroform:isoamyl alcohol (24:1). After the centrifugation,

the top layer was transferred again to a new tube.

DNA precipitation was done by adding 1/10 volume of NaOAc 3M, pH 5.3 and 2

volume of 99% ethanol. After being gently inverted, the tube was stored at -20ºC for 1

hour prior to be rotated at 14.000 rpm for 10 minutes. The DNA pellet was washed in 70%

ethanol followed by air drying. The DNA was resuspended in 100 μl of TE buffer,

incubated at 65ºC for 1 hour and ready for downstream applications.

4.3.5. Determination of DNA yield

The DNA concentration was determined by spectrofluorometry. Relative fluorescence

was measured by using a Shimadzu Fluorospectrophotometer (RF1501). The

spectrofluorometric method was performed using Picogreen dye specifically for dsDNA

(Invitrogen) and measured at 480nm excitation and 520nm emission.



4.3.5.1. Standard curve 2 μg/ml of Lambda DNA (Invitrogen) was used as working solution to create a

standard curve. A seven-point standard curve (0; 5; 10; 15; 20; 25 and 30 ng/μl) was

prepared by diluting the working solution in the same way as the experimental sample

(Table 4-1). After incubation for 5 minutes at room temperature and protected from light,

samples were measured. The fluorescence values were used to generate the standard curve

(Figure 4-2) of relative fluorescence versus the DNA concentration of the samples as

follows:

y = ax + b, where “y” is DNA concentration while “x” is relative fluorescence (RF)

Table 4-1. Protocol used to prepare the standard curve

DNA Conc. (ng/ml)

2 μg/ml λDNA Stock (μL)

TE-buffer 1X (μL)

PicoGreen® dsDNA Reagent (μL)

Total Volume (μL)

0 (blank) 0 1998 2 2000 5 5 1993 2 200010 10 1988 2 2000 15 15 1983 2 200020 20 1978 2 2000 25 25 1973 2 200030 30 1968 2 2000

Ex : 480nm Em : 520nm

Y = 20.592X + 9.1132R2 = 0.9993

0

100

200

300

400

500

600

700

0 5 10 15 20 25 30 35DNA (ng/ml)

Rel

ativ

e Fl

uoro

scen

ce

Figure 4-2. An example of standard curve for spectrofluoromric measurement



4.3.5.2. Sample Analysis

One μl of experimental DNA solution was diluted in 1997 μl TE buffer 1X to a final

volume of 1998 μl in a disposable cuvette (ROTH; Art.8128). 2 μl of PicoGreen® dsDNA

quantitation reagent was added to each sample and the samples were then incubated for 2

to 5 minutes at room temperature. The relative fluorescence was measured by using

instrument parameters that correspond with those used for generating the standard curve.

The DNA concentration of the sample was calculated from the DNA standard curve

through the following formula:

DNA (ng/μl) = [(RF-b)/a] x 2; where RF is relative fluorescence

4.3.6. Evaluation of DNA purity

The DNA purity was estimated by spectrophotometry. A NanoDrop ND-1000

spectrophotometer (Isogen) was used to calculate the ratio of absorbance at 260 and 280

nm. The analysis was carried out according to the manufacturer’s instruction (NanoDrop,

2007). 1 μl sample was pipetted onto the lower pedestal/the receiving fiber (Figure 4-3).

The integrated software measures the DNA concentration and the ratio of A260/280.

(a) (b) (c) (d)

Figure 4-3. The application of NanoDrop (a-c) and its typical data output (d)

In order to evaluate DNA integrity, including degraded DNA and contaminants, the

DNA isolates were checked by agarose GE. 1% agarose (Promega) was prepared in 1X

TAE buffer with Gel Red staining dye (Molecular Probes). For a 20x10 cm2 gel, 1 g of

agarose was diluted in 67 ml of TAE buffer 1.5X and 33 ml of GelRed in a 500 ml

Erlenmeyer flask. The mixture was heated in a microwave for +5 minutes until the agarose

was completely dissolved. After cooling down for a few minutes, the gel was poured into

the gel mold with casting comb in place. The gel was allowed to cast for 15-20 minutes,

placed in the chamber of Midicell® Primo EC-330 (Thermo) (Figure 4-4). The reservoir

was filled with 1X TAE buffer until the buffer just covered the gel. 5 μl of samples were

prepared and to each of them was added one drop of blue-orange loading dye (Promega). A

Sampling arm Lower measurement pedestal

upper measurement pedestal



Smart Ladder 1800 (Eurogentec) was used as a 1 kb DNA marker. The gel was

electrophoresed for 1 h at 66 V until the orange dye reached approximately 3/4 of the gel.

DNA fragments were visualized on a Consort UV transilluminator at 254 nm and captured

by Edas-290 digital camera (Kodak).

A. Thermal cycler B. Electrophoresis chamber C. UV illuminator

Figure 4-4. Apparatus for PCR applications

4.3.7. PCR assay

A PCR assay was employed to evaluate the efficiency of DNA isolate to amplify the

targeted fragment. The CytBL1/CytBH primer couple was used to generate ~357bp of the

cyt b gene. The reaction was performed in a final volume of 20μl. 10μl of Jump Start RED

Taq Ready Mix (Sigma P-0982) was used to obtain 10mM Tris-HCl pH 8.3, 50mM KCl,

2mM MgCl2, 0.2mM of each dNTP, and 0.03U/μl Taq DNA polymerase. 2 μl of each

primer (10mM) and serial dilution (5 and 20 ng/20μl) of DNA templates were added

following the instruction as described in Table 4-2. Amplification was carried out in a

PCRexpress thermal cycler (Hybaid). The PCR conditions included initiation (94oC for 5

min), 35 cycles of amplification (94oC for 30 sec; 50oC for 30 sec; 72oC for 1 min) and

final extension (72oC for 5 min).

Table 4-2. Composition of PCR reaction

Reagent Volume (μl) Final concentration

2x JumpStart REDTaq ReadyMix 10 μl 1x Forward primer (10 mM) 2 μl 1 μM Reverse primer (10 mM) 2 μl 1 μM DNA template variable 5 and 20 ng PCR H2O variable - Total volume 20 μl -



4.3.8. Visualization of PCR product

Amplified PCR products were analyzed by gel electrophoresis on 2% (w/v) agarose

(Promega). The preparation of the gel and visualization of the result was as described in

Section 4.3.6. (see page 35). 5 μl aliquot of amplified products was loaded on the gel

together with a 100 bp 1700 DNA ladder (Eurogentec) as the marker.

4.4. Optimization and validation of a universal PCR protocol 4.4.1. Primer design

At first, the primer couple of CytBL1/CytBH was evaluated and optimized to

amplify the selected samples. This primer couple has widely been used to amplify ~357bp

of the cyt b of various species (Kocher et al., 1989; Bartlett & Davidson, 1991 & 1992;

Céspedes et al., 1998; Richardson et al., 2006). Since this primer was not optimal for

crustacean, mollusc, and some fishes, degenerate primers were designed by introducing

some wobbles. The primers were designed by means of Oligo software following a number

of rules for primer design (Qiagen, 2005; Roche, 2006).

Alternatively, other universal primers (UCYTB) were also validated to amplify

~410bp of the cyt b gene. The primers were designed based on those described by Meritt et

al. (1998). Table 4-3 describes the properties of the primers used in this study. In addition,

the scheme of targeted fragments created by the validated primers is drawn in Figure 4-5.



(5’) 427 827/830/833 (3’)

2 &,6 9 - 12 ~ 357bp ~ 410bp 1, 3, 4, 5 7 & 8

(3’) 70 421/424 (5’)

Figure 4-5. The scheme of targeted fragments generated by the evaluated primers 4.4.2. PCR assay

The PCR reaction was performed in a final volume of 20 μl as described in Section

4.3.7. (see page 36). The PCR conditions were adjusted depending on the set of primers as

described in Table 4-4.

Table 4-4. PCR condition of the different primer sets

Primer PCR condition CytBL1 CytBH CytBL1A CytBL1B CytBL1C CytBHW

Initiation Denaturation Annealing Extension Final extension

94oC; 5 minutes; 1 x 94oC; 30 seconds 45-60oC; 30 seconds 35 cycles 72oC; 1 minute 72oC; 5 minutes; 1 x

UCYTB151BF UCYTB152BF UCYTB270B UCYTB271R UCYTB272BR UCYTB273R

Initiation Denaturation Annealing Extension Final extension

94oC; 4 minutes; 1 x 94oC; 1 minute 45-60oC; 1 minute 35 cycles 72oC; 2 minute 72oC; 5 minutes; 1 x

Amplified PCR products were analyzed by gel electrophoresis on 2% (w/v) agarose

(Promega). The preparation of the gel and visualization of the result was as described in

Section 4.3.6. (see page 35). 5 μl aliquot of amplified products was loaded onto the gel

together with a 100 bp 1700 DNA ladder (Eurogentec) as the marker.

4.4.3. Direct sequencing of PCR product

Direct sequencing was attempted to confirm whether the designed/validated primers

amplify the targeted fragments. Sequence analysis was carried out using a 4 color dye (Big

Dye, Applied Biosystems) and performed in an ABI 3730xl DNA analyzer at AGOWA

GmbH Berlin, Germany. Prior to the sequencing, PCR products were purified using the

High Pure PCR Product Purification Kit (Roche Applied Sci. Mannheim, Germany)

according to the manufacturer’s instructions.



Sequence chromatograms were viewed and evaluated by using ChromasPro and

BioEdit software (Figure 4-6). Both forward and reverse sequences were edited by

trimming out the ambiguous bases. The selected sequences were then assembled to analyze

the overlapping bases.

Figure 4-6. Software for sequence analysis: ChromasPro (left) and BioEdit (right)

4.4.4. Real Time PCR application

Prior to the RT-PCR amplification, all reagents (2x QuantiTect SYBR Green RT-

PCR Master Mix and primers) were thawn. The master mix was prepared to a final volume

of 20 μl according to the manufacturer’s instructions (Table 4-5). Into each microplate

well, 15 ul of the mix solution were filled. 5 μl of DNA template (final concentration of

10ng/20μl) was added to each well, except the blank. The microplate was centrifuged for 3

min at 3000 rpm (Sigma 3-18K; Sartorius). The RT-PCR amplification was carried out in a

LightCycler® 480 (Roche) at the conditions as described in Table 4-6. The melting curve

was determined from the thermal profile observed for 30 seconds after elongation (60-90ºC

with ramp rate 2.2-4.4ºC/s). The melting points (Tm) generated from the melting curve

(Figure 4-7) were used to discriminate between closely-related species.

Table 4-5. Reaction components of RT-PCR amplification

Component Volume/reaction Final concentration SYBR Green RT-PCR Master Mix 2x 10μl 1x Forward and reverse primer 10μM 1μl 0.5μM DNA template 5μl 10ng PCR grade H2O 3μl To make final volume Final volume 20 μl 1 x Reaction Mix



Table 4-6. Thermal profile of RT-PCR amplification

Step Time Temperature(oC) Remark

Initial activation 10 min 95 Ramp rate 4.4ºC/s Denaturation 10 sec 95 Ramp rate 4.4ºC/s

Annealing 10-20 sec 45-60 Depends on the melting temperature Ramp rate 2.2ºC/s

Elongation 30 sec 72 Ramp rate 4.4ºC/s

Cycle number 30-40 cycles Depends on the length of the targeted fragment

Figure 4-7. Typical results of RT-PCR: annealing curve (left) and melting curve (right)

4.5. Data analysis

The similarity index of selected samples based on sequence profile was compared to

the references/libraries obtained via GenBank and FishTrace by using BLAST analysis

(Figure 4-8).

Figure 4-8. An example of BLAST analysis obtained via GenBank (left) and FishTrace (right)



CHAPTER VII CONCLUSIONS

Despite yielding lower DNA concentration, the commercial kit produced better

quality of DNA isolate compared to the classical methods, performed by higher efficiency

in PCR amplification. In addition, commercial kits offer simpler procedure and avoid the

use of harmful reagents in comparison to classical methods. In the classical methods

evaluated in this study, 4 M urea in combination with 0.5% SDS (Wasko et al., 2003) or

1% SDS without urea (Hsieh et al., 2005) proved to be the optimal lysis solution for fish

and seafood products.

Typically, the CytBL1/CytBH primer couple worked effectively on most fish (except

S. maximus, M. kitt, and sand lances/smelt fish). In contrast, these primers showed low

efficiency in amplifying crustacean and molluscs. Adjustment of Mg2+ concentration did

not affect significantly on annealing efficiency of C. crangon and M. edulis. Meanwhile,

the degenerate primer couple CytBL1C/CytBHW effectively amplified most molluscs and

crustaceans, except C. crangon and I. argentinus. This primer couple is promising to be

employed universally for species identification of crustaceans and moluscan taxa. In

addition, UCYTB151BF/UCYTB271R and UCYTB152BF/ UCYTB271R were successful

to amplify all fish, crustacean and mollusc examined in this study. Hence, these primers

can be considered as the first universal primers applicable for fish, crustaceans, and

molluscs. However, further trials on a wider variety of species, including species with

unusual mtDNA character such as Pectenidae, are necessary to ascertain whether these

primers are universally effective to amplify the majority of fish and seafood products.

Sequence analysis proved that all validated primer couples were successful in

amplifying the expected DNA fragments (~357bp and ~410bp) of cyt b region. BLAST

analysis performed similarity indexes against the libraries of 15 selected samples varying

between 92% and 100%. Sequencing was also successful to differentiate the 3 unknown

sand lances/smelt fish and 2 unknown shrimps. Additionally, RT-PCR was applicable to

differentiate between tuna and cod, 2 ray fish (R. clavata and M. senta), 2 crustaceans (P.

monodon and P. latisulcatus), and 2 mussels (M. edulis and Perna canaliculus), but failed

to discriminate among smelt fish. In order to increase the RT-PCR sensitivity,

amplification using HRM Mix is suggested.



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LIST OF ABBREVIATIONS

AFLP: Amplified Fragment Length Polymorphism

ATP: Adenosine Triphosphate

BLAST: Basic Local Alignment Search Tool

BOLD: Barcode of Life Data System

COI: Cytochrome oxidase Sub Unit I

CTAB: Cetyltrimethyl Ammonium Bromide

Cyt b: Cytochrome b

ddNTP’s: Dideoxynucleotides

DGGE: Denaturing Gradient Gel Electrophoresis

ELISA: Enzyme-Linked ImmunoSorbent Assay

EMBL: European Molecular Biology Laboratory

FADH2 : Reduced Flavin-Adenine-Dinucleotide

FISH-BOL: Fish Barcode of Life Initiative

GE: Gel Electrophoresis

IEF: Isoelectric Focusing

MALDI-TOF-MS: Matrix Assisted Laser Desorption/Ionization–Time of Flight–Mass

Spectrometry

NCBI: National Center for Biotechnology Information

mtDNA: Mitochondrial DNA

NADH: Reduced Nicotinamide-Adenine-Dinucleotide

nDNA: Nuclear DNA

NMR: Nuclear Magnetic Resonance

PCR: Polymerase Chain Reaction

PFGE: Pulse-Field Gel Electrophoresis

RAPD: Random Amplified Polymorphic DNA

RFID: Radio Frequency Identification

RFLP: Restriction Fragment Length Polymorphism

RT-PCR: Real Time Polymerase Chain Reaction

SDS-PAGE: Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

SSCP: Single Strand Conformation Polymorphism

TGGE: Temperature Gradient Gel Electrophoresis



LIST OF WEBSITES

BioEdit: http://www.mbio.ncsu.edu/BioEdit/bioedit.html

BOLD: http://www.barcodinglife.org

ChromasPro: http://www.technelysium.com.au/ChromasPro.html

DNAsoftware: http://www.dnasoftware.com/Science/primerdesign.html

EMBL (EMBL-bank): http://www.ebi.ac.uk/embl/

ExPASy: http://www.expasy.org/

FISH-BOL: http://www.fishbol.org

FishTrace: http://www.fishtrace.org

Mascot: http://matrixscience.com/

MitoFish: http://mitofish.ori.u-tokyo.ac.jp/

NCBI (GenBank): http://www.ncbi.nlm.nih.gov/

OligoDesign: http://www.invitrogen.com/oligos

PeptideMass: http://www.expasy.ch/tools/peptide-mass.html

PremierBiosoft: http://www.premierbiosoft.com/primerdesign/index.html

PrimerDesign: http://www.primerdesign.co.uk/Biosearch



APPENDIX I. List of samples used in this study

1. Thunnus sp 2. Eutrigla gurnardus

3. Gadus morhua 4. Trisopterus luscus

5. Solea solea 6. Microstomus kitt 7. Raja clavata 8. Malacoraja senta

11. Unknown smelts

12. Crustaceans

14. Perna canalicullus 15. Sea mussels

9. Ammodytes tobianus

10. Hyperoplus lanceolatus

13. Illex argentinus

P. monodon M. rosenbergii

P. latisulcatus

M. edulis

C. crangon

Mussel-T1 Mussel-120 Mussel-2251



APPENDIX II. List of chemicals, solutions, and equipment

Table II-A. List of chemicals and reagents

Chemical Company Ordering number Ethanol p.a. Merck 8.18760.2500 EDTA-Na2 dihydrate BDH 443885J Tris Base Promega A5135 Nuclei Lysis Solution Promega A7943 Proteinase K recombinant PCR Roche 3115879 RNAase Roche 10109169001 Protein Precipitation Solution Promega A7953 Isopropanol p.a. Serva 39559.02 NaOH p.a. BDH 102525P HCl 37% Merck 1.00317.2501 PCR-H2O18.2 MΩ cm Sartorius Arium

Table II-B. List of solutions

EDTA 0.5M pH 8.0

93g EDTA-Na2 dihydrate (Mw: 372.2) Add 350ml PCR-H2O, add 10N NaOH until pH 8.0 Add PCR-H2O until 500ml and sterilize for 20 minute at 121°C (1 bar)

NaOH 10N Add 40g NaOH with 100ml PCR-H2O 500ml and sterilize for 20 minute at 121°C (1 bar)

Tris-HCl 1M, pH 7.4 30.29g Tris Base (MM: 121.14) with 150ml PCR-H2O in a 250ml volumetric flask. Add HCl 37% until pH 7.4, add with H2O until 250ml and sterilize for 20 minute at 121°C (1 bar)

TE-buffer 1X 10mM Tris-HCl, pH 7.4 1mM EDTA, pH 8.0

Mix 10ml Tris-HCl 1M, pH 7.4 and 2ml EDTA 0.5M, pH 8.0 then add with PCR-H2O until 1 lt. Sterilize for 20 minute at 121°C (1 bar).

RNAse Solution 4mg/ml Weigh 100mg RNase A in 25ml PCR-H2O in a 25 ml volumetric flask

Proteinase K solution 20 mg/ml Weigh 20mg Proteinase K in a 1.5ml microcentrifuge tube. Add 1 ml sterilized PCR water and vortex

TNES buffer with 0.5 or 1% SDS 10 mM Tris HCl pH 7.5, 125mM NaCl, 10mM EDTA, 0.5 or 1% SDS

Urea 4 or 6 M 48.05 or 72.08g in 200ml TNES buffer

Table II-C. List of equipment

Equipment Specification Company Plastic tubes 1.5-2.5 ml ROTHMicro centrifuge Sigma 1-14 Sigma Filter/fin tip 10 – 1,000 μl Molecular BioProducts Analytical balance - Sartorius, Mettler Water bath 20-110ºC Memmert Vortex Mini vortexer-TM1 Techmatic

PCR Protocol for Fish and Seafood Authentication - MSc Thesis

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