Amplified Fragment Length Polymorphism AFLP, Frequent Cutter and Rare Cutter, Facilitate designing of Primers for Selection, Oligonucleotide Adapters

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Home >> Biotechnology and Genomics >> DNA-Based Molecular Markers in Biotechnology >>Amplified Fragment Lenght Polymorphism Aflp

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Amplified Fragment Length Polymorphism (AFLP) and its modified forms.

AFLP is based on PCR amplification of a set of restriction fragments, selected from a pool of fragments, that are generated due to digestion with a pair of specific restriction enzymes,

one of them being a frequent cutter (e.g. Msel), and the other being a rare cutter (e.g. EcoRI). To facilitate designing of primers for selection of restriction fragments, oligonucleotide adapters, few base pairs (-20) long are ligated at the ends of these DNA fragments.

With the help of these ligated adapters, the number of DNA fragments to be amplified can be restricted, since the primers are designed to bind to the following sequence: ligated adapters + the restriction site of the enzyme used for digestion + 1-3 selective bases (chosen randomly).

This method generates a large number of bands (but not too many to become difficult to score,

Amplification Profiles inWheat Genotypes Obtained using A. AFLP

Amplification Profiles in Wheat Genotypes Obtained Using AFLP

Amplificatiojn Profiles in Wheat Genotypes Obtained Using B. SAMPL

Amplification Profiles in Wheat Genotypes Obtained using b SAMPL

representing the amplified products from selected restriction fragments, thus facilitating the, detection' of polymorphism.

It has been shown that in AFLP, a single primer combination detects up to 8 times more polymorphic bands and up to 16 times more loci, when compared with RFLP.

A comparison of different molecular marker techniques (RFLP, RAPD, SSR and AFLP) also demonstrated that AFLP is the most efficient technique for detecting polymorphism.

Consequently, AFLP markers have been used for a variety of purposes including the preparation of molecular maps.

The technique of AFLP has been variously modified, where an AFLP primer with 3 selective nucleotides is used either in combination with a microsatellite primer (selective amplification of microsatellite polymorphic loci = SAMPL) or in combination with a retroposon based primer (sequence specific amplification polymorphism = S-SAP).

Generally, SAMPL primer (18-20 nucleotides long) is based on sequences of two different adjacent SSRs and an associated intervening sequence, known to be found in compound repeats (as shown in corn and soybean).

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Rarely, SAMPL primers each based on a single SSR (with or without a small non- microsatellite sequence at 5' end for anchoring) have also been used.

In fact, many different combinations of restriction endonucleases and primers can be used for SAMPL, allowing for the detection of nearly limitless SSR-based polymorphisms in a crop like wheat,

where AFLP produces too many crowded bands to score, SAMPL gives relatively fewer bands but resolving higher level of polymorphism, thus making this technique more rewarding and user friendly.

In several of the above studies, it has also been shown that SAMPL (presumably also S-SAP) markers, like AFLP, are also often dominant, but since in case of SAMPL,

the polymorphism can one often be due to SSR length polymorphism, a fraction of SAMPLE markers can also be co-dominant. Independent data (segregation, and sequence information) may be needed to confirm the co-dominant nature of these markers.

A polymorphic band from a SAMPL or S-SAP fingerprint gel can also be converted into a conventional, single locus PCR-based marker by cloning and sequencing.

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Amplifed Fragment Length Polymorphism (AFLP) Single Strand Conformation Polymorphism (SSCP) Molecular Markers based on PCR Followed by Hybridization Oligonucleotide Fingerprinting Using RAPD/MP-PCR Framents Sequence based Molecular Markers Single Nucleotide Polymorphisms (SNPs) Gel-based Assays of PCR Products for SNP Detection Non-Gel based Assays of PCR Products for SNP Detection Taqman Assay Use of Molecular Beacon Oligonucleotide Ligation Assay (OLA) DNA Chip/Microarray Dynamic Allele-specific Hybridization Minisequencing Pyrosequencing for SNP Genotyping Temperature Modulated Heteroduplex Analysis TMHA Using DHPLC Wave^TM System A Non PCR Invasive Clevage Assay and Maldi TOF-MS for SNP Detection Comparison of Different Types of Molecular Markers High Throughput Approach in Molecular Marker Technology Comparison of General Features of Different types of Molecular and their use Comparison of General Features of Different types of Molecular and their use