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Polymerase Chain Reaction: A Robust Technology for Crop Improvement Vivekanand Tiwari*, Kiran Patel, Viral B. Mandaliya , and Sujit Kumar Bishi

Directorate of Groundnut Research, PB-05, Ivnagar Road, Junagadh-362 001, Gujarat *Email of corresponding author: vivekcas805@gmail.com

Introduction

In 1983, Karry Mullis and co-workers have developed PCR technique to replicate fragments of DNA by semi-conservative method but under in-vitro condition. The introduction of thermostable Taq DNA polymerase from Thermus aquaticus has facilitated automation of this technology (Saiki et al., 1988). The technique was patented by Kary Mullis at Cetus Corporation, where he invented. The use of this invention for crop improvement was widely accepted in the areas of research like detection of plant pathogens, development of genetic or physical map of crop genome, cloning and characterization of existing or new trait responsive genes and their expression pattern analysis, genetic diversity analysis and marker assisted breeding for development of new crop varieties. Although, PCR has become a routine tool in molecular biology research for crop improvement studies, but still there are few important points should be taken care while performing a PCR. Different modifications in the components and reaction conditions have been adopted according to the applications or aim of the experiments for successful PCR reaction. This articles focus on the basic principle of PCR, what are the precautions should be taken care and application of PCR for crop improvements.

Principle of PCR The principle of a PCR reaction can be generalized as the, in-vitro replication of small fragment of template DNA upto a desired size (< 10 kb). The DNA product formed after PCR are known as amplicons. Amplicons are increased in number exponentially after completion of each cycle. It includes separation of two opposite DNA strand by heat denaturation instead of enzymatic unwinding occurs in cells. A pair of small single stranded DNA fragments

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Polymerase Chain Reaction (PCR) is widely used molecular biology research tools discovered by Kary Mullis in 1983. In this technology, with use of thermostable Taq DNA polymerase a fragment of DNA was transcribed in-vitro. The amplification was carried out using small fragment of DNA complementary to both ends of the DNA to be amplified and called as primer. Primers were designed carefully to avoid non-specific amplification. This technology widely used in crop improvement programs like development of genetic or physical map of crop genome, cloning and characterization of existing or new trait responsive genes and their expression pattern analysis, genetic diversity analysis and marker assisted breeding for development of new crop varieties.

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(primer) in opposite direction to each other and complementary to the template DNA are used in the reaction which acts as substrate for addition of a series of nucleotide by phosphodiester bonding. And this is facilitated by use of a thermostable Taq DNA polymerase in the reaction. The addition of nucleotide occurs at the 3’-OH group of the primer.

In practical, all reaction components are assembled on ice and added one by one in a single reaction tube. Generally, polymerase is added at the last and final reaction volume is made by doubled distilled water or MiliQ water. Immediately reactions are transferred to thermocycler machine for starting the reactions. The following three basic steps govern the PCR reaction in DNA amplification as shown in the Figure 1. An ideal PCR reaction involves three steps; denaturation, primer annealing and extension. 1. Denaturation Temperature and Duration: Initial denaturation at 95°C for 3-10 minutes

is recommended prior to PCR cycling to fully denature the DNA. Subsequently, 10-30 second of denaturation at same temperature is sufficient in start of cycle.

2. Annealing Temperature and Duration: At this temperature both primers anneal to complementary sequence at the template DNA. The optimum annealing temperature is 5 °C lower temperature than the primer with lowest melting temperature (Tm) and difference between Tm of both primers should not be more than 5 °C. Primers are designed so that the annealing temperature fall in the range of 50-60 °C and annealing times are 15-30 seconds.

3. Extension Temperature and Duration: Extensions are normally performed at 72°C. At this temperature, nucleotides are added at the 3’-OH end of both primes by Taq DNA polymerase and extended upto the desired length. Generally, for extension of 1000 bases one minute is sufficient (e.g. 2 minutes for a 2 kb product). For products less than 1 kb, 45-60 seconds is advised. Products greater than 3 kb, practically it is difficult to amplify by using normal Taq DNA polymerase. Special kind of high fidelity polymerase enzymes are required.

Figure 1: Three different steps Denaturation, Annealing and Extention, of a PCR reaction. F and R during the annealing steps represent forward and reverse primers.

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Table 1: Optimization of PCR Particulars/ PCR Components

DNA template

Reaction buffer

dNTPs MgCl2 (Mg+2)

Primers Taq DNA Polymerase

Enhancer

Role Provide the complementary site for amplification

Maintains pH, ionic strength, cofactors etc.

Building blocks of amplification

Act as co-factor for enzyme

Provide the 3’ OH group for amplification

Enzyme to amplify the template

Minimizes secondary structures in the DNA template and primer

Optimum concentration/ reaction (25 µl)

50- 100 ng (eukaryotes) 10- 25 ng (prokaryotes)

As suggested by supplier, preferably 1X

100- 200 µM of each nucleotides

1-4 mM 5 - 20 pmol

1 - 2 Unit

Varies

Higher concentration

Non-specific amplification

Inhibition of enzyme

Lowers enzyme activity by trapping Mg+2 ions

Increase the non-specific binding of primer and decreased specificity of the reaction

Non-specific amplification

Non-specific amplification

Non-specific amplification

Lower concentration

No or low amplification

Loss of enzymatic activity

Truncated amplification

Reduced Taq polymerase activity

Reduced amplicon number

Low PCR products

Not affecting too much

Table 2: Inhibitors of PCR reactions Inhibitors Concentration

SDS >0.005% (w/v) Phenol >0.2% (v/v) Ethanol >1% (v/v)

Isopropanol >1% (v/v) sodium acetate >5 mM sodium chloride >25 mM

EDTA >0.5 mM

(Sources: Kramer, et al., 2001, Sambrook and Russel, 2001, Pavlov, et al., 2004, and Joshi and Deshpande, 2010)

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Components of PCR For a successful completion of PCR reaction, the optimum concentrations of its components are required. These components and their optimum concentrations are listed in the following Table 1. The primers were designed and custom synthesized from the commercial industries like Sigma, IDT, Europhin etc. While designing primer, there are few facts which always should be kept in mind.

Must keep in mind while designing primers • Optimal annealing Temperature should be preferably about 55 to 65°C • The GC-contents of each primer should be between 40-60%. • Difference of calculated Tm for both primers should not more than 5°C. • The annealing temperature in PCR reaction should be 5°C lower than that of primer with

lower calculated Tm. • The self-complementary hairpins forming nucleotide in a single primer should not >3

and number of dimer forming nucleotides between primer pair should not >7. • The 3' terminus of primer must end with C or G nucleotide which minimizes non-

specific annealing. Also the GC content in 5 nucleotides at 3’ terminus should not exceeds 60%.

Enhancer and inhibitors of PCR There are several PCR additives rarely used as PCR enhancer such like dimethyl sulfoxide (DMSO), glycerol, formamide, betaine, and/or proprietary compounds. These compounds will enhance the PCR amplification efficiency. Similarly, various compounds in plant material and food products can be co-extracted with genomic DNA and inhibit the PCR. This may lead to false-negative results because of failure of the PCR. The type of inhibitors and their concentration are listed in Table 2.

Applications of PCR technology in crop improvement 1. To increase the number of very few number of DNA template. 2. Isolation of a orthologous gene sequence by using degenerate primers designed from

closely related plant species gene sequence alignments. 3. Amplification and isolation of potential trait responsive genes from crops and their

transcript expression pattern analysis under different stress conditions. 4. Synthesis of complementary DNA (cDNA) from RNA isolated from the crop using

modified process of PCR called Reverse-transcriptase PCR. 5. Amplification of gene from cDNA. 6. PCR is used for DNA sequencing to determine unknown PCR-amplified sequences,

which helps in gene discovery. 7. PCR remains as an integral tool for cloning, vector construction, and transformant

identification into bacterial cells. 8. Identification of genetically modified crops for the presence of transgene using PCR. 9. PCR amplification and sequencing for marker genes like 16S and 18S ribosomal RNA

genes in prokaryotes and eukaryotes, respectively for phylogenetic analysis. 10. Screening and differentiation of diseased with viral pathogen and healthy plants using

viral gene specific primers. 11. Screening and characterization of genotypes during marker assisted breeding for

development of new varieties in different crop plants use PCR. 12. Seed quality control (Lipp et al. 2005): Commodity and food companies, as well as

third-party diagnostic testing companies, rely on PCR technology to verify the presence or absence of transgene material in a product or to quantify the amount of transgene material present in a product. The copy number of transgene and zygosity is etermined by quantitative PCR technology.

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Conclusion

Development of advanced technologies has transformed scientific research in the ways that was unpredictable just a half century ago. Polymerase Chain Reaction (PCR) is one among those inventions that has completely revolutionized the molecular biology research either in animal science or plant science. References

Joshi M and Deshpande JD. 2010. Polymerase Chain Reaction: methods, principles and application. International Journal of Biomedical Research, 2: 81-97.

Kramer MF and Coen DM. 2001. Enzymatic amplification of DNA by PCR: standard procedures and optimization. Current Protocols in Molecular Biology, 15: 1.1-1.14.

Lipp M, Shillito R, Giroux R, Spiegelhalter F, Charlton S, Pinero D and Song P. 2005. Polymerase Chain Reaction technology as analytical tool in agricultural biotechnology. Journal of AOAC International, 88: 136-55.

Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI. 2004. Recent developments in the optimization of thermostable DNA polymerases for efficient applications. Trends in Biotechnology 22: 253–260.

Saiki R, Gelfand D, Stoffel S, Scharf S, Higuchi R, Horn G, Mullis K and Erlich H. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491.

Sambrook J and Russel DW. 2001. Molecular Cloning: A laboratory manual (3rd ed.). Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. ISBN 0-879-69576-5. Chapter 8: In vitro Amplification of DNA by the Polymerase Chain Reaction, PP8.1-8.24

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