AMPLIFIED FRAGMENT LENGTH

POLYMORPHISM

SUBMITTED TO SUBMITTED BY
MALAVIKA M.R
DR.BINDHUMOL ISMAIL
Roll. No: 06 II MSc
DEPT. OF BIOTECHNOLOGY
DEPT. OF BIOTECHNOLOGY
 AFLP
• A molecular marker is a DNA sequence in the genome which can be located and identified.

• As a result of genetic alterations such as mutations, the base composition at a particular location of the
genome maybe different.

• The differences are collectively known as polymorphisms.

• Molecular markers are of two types .

1. Based on nucleic acid hybridization

2. Based on PCR amplification.

• PCR based markers can be divided into two types.

A. Locus , non specific markers :- RAPD, AFLP

B. Locus, specific markers :- SSR and SNP

• Amplified Fragment Length Polymorphism is a highly sensitive method for detecting
polymorphisms in DNA.

• The technique was originally described by Zabeau and Vos in 1993 and by Vos and Kuiper in 1997.

• This method is based on PCR amplification of selected restriction fragments obtained after
digestion of total genomic DNA with restriction endonucleases .

• Polymorphisms are then detected by studying differences in length of the amplified fragments
separated using Gel electrophoresis.

• AFLP is a technique involving combination of RFLP and RAPD

• The process consists of four steps:-
a) Restriction digestion of genomic DNA

b) Ligation of adapters

c) PCR amplification

d) Gel electrophoresis and analysis

I. Preparing the AFLP Template

• After the genomic DNA is obtained, it is digested with a pair of restriction enzymes, often MseI and
EcoRI .

• MseI recognizes 5′-TTAA-3′ 4 base pair recognition site and cleaves after the first 5′-T,
whereas EcoRI recognizes 5′-GAATTC-3′ 6 base pair recognition site and cleaves after the 5′-
G.

• The genomic DNA is typically incubated with these enzymes for a period long enough to permit
complete digestion of the DNA into restriction fragments.

• MseI and EcoRI generate DNA fragments with 5′ overhangs (5′-TA-3′ and 5′-AATT-3′,
respectively) that are distinct from each other.
2. Ligation Reaction with Restriction Fragments and Adaptors
• MseI adaptor and an EcoRI adaptor, each of which is a short, double-stranded
DNA molecule with a 5′ overhang that is complementary to the 5′ overhang generated by
MseI (5′-TA-3′) and EcoRI (5′-AATT-3′), respectively.

• Rather than containing a 3′-A after the 5′-TA-3′ overhang, the MseI adaptor contains a
3′-C, which destroys the MseI recognition site once ligated.

• Similarly, rather than containing a 3′-C after the 5′-AATT-3′ overhang, the EcoRI
adaptor contains a 3′-G, which destroys the EcoRI recognition site once ligated.

• The MseI and EcoRI adaptors each contain their own specific DNA sequence, typically between
19 and 22 base pairs in length, that is located upstream of their respective 5′ overhangs.
3. Selective PCR Amplification

• If PCR reaction is carried out using primers corresponding to the MseI and EcoRI adaptor
sequences, they would amplify every single genomic DNA fragment.
• Therefore be faced with an indecipherable set of DNA fragments.
• Thus, in order to selectively amplify a smaller number of genomic DNA fragments primer sets
that are complementary to the MseI or EcoRI adaptor sequences are used.
• To be more specific , a small number of base pairs, called selective nucleotides are added, at the
end of each primer.
• This permits the amplification of a subset of genomic DNA fragments.
• The more complex the genome being investigated, the more selective nucleotides researchers
add to the primers.
4. Electrophoretic Separation of Amplified DNA Fragments
• To identify and characterize the set of DNA fragments produced in a given PCR reaction ,one of
the two PCR primers is radioactively or fluorescently labeled (typically the EcoRI primer), which
leads to the production of a labeled PCR product that can be easily detected.

• Electrophoresis is used to separate the DNA fragments based on their size and overall negative
charge, large fragments will migrate more slowly, and small fragments will migrate more quickly
when exposed to an electrical field.

• Polymorphisms are detected as the presence or absence of bands mainly due to mutation ,
insertion or deletion within amplified restriction fragments.
APPLICATIONS
• Used to indicate whether two organisms are members of the same species.

• Genetic variations

• Criminal investigation and paternity tests.

• Track pathogenic outbreak

• Gene expression fingerprints

ADVANTAGES
• Highly reproducible.

• Provides whole genome scan of polymorphism

• No prior knowledge of sequence required.

DISADVANTAGES
• Need for high amount of purified DNA

• Expensive