PRESENTED BY

AMBER HASSAN

(PhD scholar)

AZNOSTICS THE DIGNOSTIC CENTER

PRINCIPLE OF AFLP

The AFLP technique is based on the principle

of selectively amplifying a subset of restriction

fragments from a complex mixture of DNA

fragments obtained after digestion of genomic

DNA with restriction endonucleases.

PROCEDURE IN AFLP Following steps are involve in AFLP:

- Digestion

- Adaptor Ligation

- Amplification

- Electrophoresis

Two different restriction endonucleases are used in digestion. One is 4-base cutter (MseI)

and the other one is 6-base cutter (EcoRI).

MseI 5’TTAA3’

EcoRI 5’GAATTC3’

- Two different adaptors (short double stranded DNA sequences with sticky end) are

ligated to the digested fragments.

- One adaptor will complement to the Msel cut end, the other will complement to the EcoRI

cut end.

- DNA fragments with MseI-EcoRI ends with be selected as DNA template for amplication.

- two PCR primers complementary to the two adaptors are used in amplification.

- the PCR primers are labelled with radioactive or fluorescence dye for detection of DNA

bands on gels

- polyacrylamide gel is used for separating DNA bands.

- Normally, 30-100 DNA bands can be detected by AFLP on polycrylamide gel.

CHARACTERISTICS OF AFLP- dominant marker.

- DNA variation is detected by presence/absence of DNA bands due to:

a) presence/absence of restriction sites

b) additional bases (insertion) between two restriction sites are too large

ADVANTAGES- higher reproducibility compared to RAPD.

- highly polymorphic

RAPD It is a type of PCR reaction, but the segments of DNA

that are amplified are random.

RAPD creates several short primers (8–12 nucleotides), then

proceeds with the PCR using a large template of genomic

DNA, the fragments will amplify.

By resolving the resulting patterns, a semi-unique profile can

be gleaned from a RAPD reaction.

PRINCIPLE OF RAPD

RAPD is a PCR based technique for identifying geneticvariation. It involves use of single arbitrary primer in aPCR reaction, resulting in amplification of many discreteDNA. RAPD technology provides a quick and efficientscreen for DNA sequence based polymorphism at a verylarge number of loci.

RAPD is a method develop in 1990 similar to PCR.

It is different from conventional PCR as it need one primer for amplification. The size of primer is shorter(10 nucleotides) therefore less specific.

- the primers can be designed without the experimenter having any genetic information for the organism being tested.

Genomic DNA normally has complimentary sequences to RAPD primers at many locations.

The RAPD technology has provided a quick and efficient screen for DNA-sequence polymorphisms at a very large no of loci.

- Normally, a few (3-20) loci can be amplified by one single RAPD primer.

PROCEDURE Extraction of DNA

Selection of Primers:

The standard RAPD technology utilises short syntheticoligonucleotides (10 bases long) of random sequences asprimers to amplify nanogram amounts of total genomicDNA under low annealing temperatures by PCR.

PCR AmplificationThe polymerase Chain Reaction (PCR) is a relativelysimple but powerful technique that amplifies a DNAtemplate to produce multiple copies of specific DNAfragment in vitro. PCR amplification consists offollowing 3 steps:

DENATURATION

ANNEALING-

EXTENSION

Agarose Gel Electrophoresis of PCRAmplified DNA

ADVANTAGES OF RAPDMain advantages of the RAPD technology include

(i) suitability for work on anonymous genomes.

(ii)applicability to problems where only limited quantitiesof DNA are available.

(iii) efficiency and low expense

DISADVANTAGES OF RAPD

Amplification either occurs at a locus or it does not, leading to scores based on band presence or absence.

This means that homozygotes and heterozygotescannot be distinguished.Nothing is known about the identity of the amplification products unless the studies are supported by pedigree analysis.

CONCLUSION RAPD is probably the easiest and cheapest methods for

laboratory just beginning to use molecular markers. RAPD markers have found a wide range of applications in

-gene mapping,- population genetics, -molecular evolutionary genetics - plant and animal breeding.

This is mainly due to the speed, cost and efficiency of the RAPD technique to generate large numbers of markers in a short period comparedwith previous methods.

RESTRICTION FRAFMENT LENGTH POLYMORPHISM

RFLP –Restriction Fragment Length Polymorphism

Restriction analysis of DNA by its

digestion with restriction endonucleases

(RE) in specific restriction sites

RESTRICTION ENDONUCLEASES Enzymes that cleave DNA molecules at specific

nucleotide sequences.

Shorter the recognition sequence, the greater the number of fragments generated.

Restriction enzymes are isolated from a wide variety of bacterial genera

For example, HindII enzyme cuts at GTGCAC or GTTAAC.

• Variation in the DNA sequence of a genome detected by breaking DNA into pieces with restriction enzymes.

• REs -recognize specific 4, 5, 6, or 8 base pair (bp) nucleotide sequences and cut DNA

• Change in DNA causes:

Gain of restriction site

Loss of restriction site

ANALYSIS TECHNIQUE fragmenting a sample of DNA by a restriction

enzyme

resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis.

Then transferred to a membrane via the Southern blot procedure.

TECHNIQUE

SOUTHERN BLOTTING A method to visualize specific segments of DNA–

usually a particular gene.

Uses radioactive probes that bind to the specific DNA segments.

STEPS• Soak gel in basic solution to separate DNA strands

• Transfer DNA on to a nylon membrane (spacing of DNA is maintained)

• Incubate with radioactive probe for specific segment

• Wash away unbound probe

• Detect probes using x-ray film autoradiograph

APPLICATIONGenotyping

Forensics

Paternity tests

Patterns in hereditary diseases

Families can find out who are at risk for the disease and who are carriers

DISADVANTAGES Large amounts of DNA required

Automation not possible

Few loci detected per assay

Need a suitable probe library

Time consuming, especially with single-copy probes

Costly and Distribution of probes to collaborating laboratories required

Moderately demanding technically

Different probe/enzyme combinations may be needed