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Uploaded byKAUSHAL SAHU
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molecular marker AFLP, and application

The document outlines the Amplified Fragment Length Polymorphism (AFLP) technique, highlighting its methodology which includes digestion, adaptor ligation, amplification, and electrophoresis. It discusses the advantages of AFLP over other methods, such as high sensitivity and reproducibility, alongside its applications in various fields like plant genetics and forensic analysis. However, it also notes some disadvantages, including cost and technical demands.

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AFLP MARKER By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )
Synopsis- • Introduction • Steps in AFLP • Advantage of AFLP • Disadvantage of AFLP • Application • Conclusion • Reference
Amplified fragment length polymorphism • AFLP method based on PCR developed in 1995 by Vos et al. - Involves the use of RFLP and RAPD techniques and universally used. - Compared with the widely used RFLP, AFLP is faster, less labour intensive and provide more information. - An additional advantage over RAPD is their reproducibility.
Amplified fragment length polymorphism • This is highly sensitive method for detecting polymorphism throughout the gnome and is becoming increasingly popular. • 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.
STEPS IN AFLP • Digestion • Adaptor Ligation • Amplification • Selective amplification • Electrophoresis • Data collection
DIGESTION • Two different restriction endonucleases are used in digestion. One is 4-base cutter (MseI) and the other one is 6-base cutter (EcoRI). GAATTC CTTAAG AATT EcoR I Mse I AATTC G T AAT TTAA
ADAPTOR LIGATION • 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. AATTC G T AAT EcoR I adapter Mse I Adapter AATTC TTAAG TTA AAT
AMPLIFICATION • 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. AATTC TTAAG TTA AAT AATTC TTAAG TTA AAT
SELECTIVE AMPLIFICATION Selective Bases - • The number of DNA bands detected by AFLP is high. It can be reduced by adding selective bases (1-3 nucleotides) at the 3’-end of the PCR primers. • One additional selective base on the primer can reduced the number of DNA bands 16 folds. • Three additional selective bases on the primer can reduce the number of DNA bands 4,000 folds.
SELECTIVE AMPLIFICATION AATTCN TTAAGN NTTA NAAT AATTCA TTAAGT GTTA CAAT AATTCAAC TTAAGTTG TTGTTA AACAAT A C AAC AAC
ELECTROPHORESIS • polyacrylamide gel is used for separating DNA bands. • Normally, 30-100 DNA bands can be detected by AFLP on polycrylamide gel.
GAATTC CTTAAG TTAA AATT EcoR I Mse I AATTC G T AAT EcoR I adapter Mse I Adapter AATTCN TTAAGN NTTA NAAT AATTCA TTAAGT GTTA CAAT AATTCAAC TTAAGTTG TTGTTA AACAAT A C AAC AAC polyacrylamide gel electrophoresis
DATA COLLECTION • Instrument for electrophoretic separation and fluorescence detection • During the electrophoresis, the instrument monitors the passage the fluorescent, 5' dye- labeled fragments through the polymer by detecting fluctuations in emitted light when the fragments migrate past a fixed laser beam. • When finished, the instrument assembles the spectral data for each sample and stores it a sample file, or saves it to the application database.
ADVANTAGE • This technique is extremely sensitive. • It has high reproducibility proving extremely proficient in revealing diversity. • It discriminates heterozygote's from when a gel scanner is used. • It permits detection of restriction fragments in any background or complexity including pooled DNA samples and colned DNA segments. • It not a simple fingerprinting technique but it can be used for mapping also.
DISADVANTAGE • It is extremely expensive and requires more DNA than is needed is RAPD. • It is technically more demanding than RAPSs as it requires experience of sequencing gels. • AFLPs are expensive to generate as silver staining, fluorescent dye or radioactivity detects the bands.
APPLICATION • To detect various polymorphisms in different genomic regions. • For the identification of genetic variation in strains or closely related species of plants, fungi, animals, and bacteria. • The AFLP technology has been used in criminal and paternity tests. • In linkage studies to generate maps for qualitative trait locus (QTL) analysis.
REFERENCE • Plant Biotechnology- H. S. chawla • Biotechnology- U. satyanarayana • Biotechnology- B. D. Singh • www.ncbi.com • www.Wikipedia.com