![]() Same will be the case for the other restriction sites. So all the fragments having the adapter part, recognition site of EcoRI and a ‘G’ will only be amplified. In the fig 2: the primer is complementary to the adaptor, the remaining part of the EcoRI recognition site and an extended ‘C’. The nucleotide chosen has to be decided on the basis of the final electrophoretic results.įig 2: Preselective Amplification in AFLP using +1 nucleotide (here C) Doing this reduces the bulk of DNA and ‘selects’ only a subset of fragments. Hence the fragments having the particular complementary base pair will only be amplified. The primer is used is complementary to the sequence of adapter, the restriction site and plus one nucleotide. In the first amplification step, that is the Preselective Amplification, a subset of the restricted fragments are only amplified. There are two levels of selectivity carried out in two different steps:- Fig 1: The steps in AFLP Only those restriction fragments will be amplified that have adapter region, the recognition site and complementary nucleotides to those ‘arbitrary nucleotides sequence’. The selectivity is achieved by designing PCR primers that anneal specifically to the adaptor, recognition site and has one to three (or even five) arbitrary chosen nucleotides at the 3′ end. The primers are designed complementary to (and they anneal to) the adaptors and restriction site.Īs the gDNA is very large and May generate a very large number of restriction fragments, selective amplification of only a subset of fragments is carried out. The restricted fragments ligated with adapters are then amplified. Their sequences are known and they flank the DNA fragments, hence used to design primers, in the next step of PCR amplification. of known sequence) linker oligonucleotides with sequence homologous to the restriction site. Adaptors are chemically synthesized (i.e. The double-stranded adaptors are then attached to the either ends of the cleaved DNA fragments (most of the times, step a an b are performed together). (b) Ligation of adaptors to ends of the digested fragments: In case of EcoR1 and Mse1, EcoR1 (6 base pair long restriction site) is a rare cutter and Mse1 is a frequent cutter (4 bps long restriction site). Both these restriction enzymes produce sticky ends (overhanging ends). Each may differ in their frequency of cutting the DNA, that is the distribution of their restriction sites in the DNA may vary.Įxample: let us consider in our case EcoR1 and Mse1. The DNA used is large DNA like genomic DNA (gDNA), hence more than one type of restriction enzymes are used. This entire procedure can be divided into four main steps: The fragments are run and visualized on denaturing polyacrylamide gels either through autoradiography or fluorescence methodologies (labelled primers) and analyzed A subset of the ligated restriction fragments is then amplified using two primers complementary to the adaptor and restriction site fragments. In this, the DNA sample is first cut using restriction enzymes, followed by ligation of complementary double stranded adaptors to the ends of the restriction fragments. The original concept was brainchild of Vos and Zabeauin 1993 and combines the principles of two different techniques namely Restriction Length Fragment Polymorphism (RFLP) and Polymerase Chain Reaction (PCR). This post we shall discuss one of the widely used technique for DNA fingerprinting.ĪFLP or Amplified fragment length polymorphism is a highly sensitive method DNA fingerprinting technique, that is, it is used for detecting polymorphisms in the given sample of DNA.
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