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- 1. SEMESTER – IV BIOTECHNOLOGY SEMINAR TOPIC : AFLP AMPLIFIED FRAGMENT LENGTH POLYMORPHISM SUBMITTED BY, P. ANUSHIYA II M.SC MICROBIOLOGY REG NO : 20201232516102 Submitted to, G. Ramanathan Assistant professor Department of microbiology
- 4. INTRODUCTION FIRST DESCRIBED BY VOS AND ZABEAU IN 1993. INVOLVES THE USE OF RFLP AND PCR TECHNIQUES AMPLIFY THE SAME GENE FROM DIFFERENT INDIVIDUAL POWERFUL APPROACH TO DETECT POLYMORPHISM
- 5. AFLP Uses restriction enzymes to digest genomic DNA Ligation of adaptors to the sticky ends of restriction fragments Amplification of selected subset of the restriction fragments (60-500 bp) Higher repeatability compared to RAPD and ISSR
- 6. AFLP Even small amount of genomic DNA can be used to produce DNA fingerprints that are highly specific to particular specices Does not require any prior of the genome sequence
- 7. AFLP Uses many of the same steps as the other markers (RFLP, SSR ,RAPD ) Includes additional steps that permit high resolution interrogation of the entire genome Yields highly specific , reproducible genotypic data
- 11. RESTRICTION NZYMES Found in bacteria Cut DNA within the molecule (End on nuclease ) Cut at sequences that are specific for each enzyme leave either blunt or sticky ends depending upon the specific enzyme .
- 12. ADAPTOR LIGATION Two different adaptors are ligated to the digested fragments one adaptor will complement the other will complement to the ECORI cut end
- 13. ADAPTOR LIGATION
- 14. AMPLIFICATION
- 15. ELECTROPHORESIS Polyacrylamide gel It detects 30-100 DNA bands
- 17. SELECTIVE BASES Added at the 3’-end of the primers 1-3 nucleotides It can reduce the number of DNA bands 1 nucleotide – up to 16 folds 3 nucleotides – up to 4000 folds
- 18. GENOTYPING If there are 2 new priming sites within 400-1600bp = amplification result = presence orabsence of amplification mostly due to SNP also deletions or insertions
- 19. 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
- 20. ADVANTAGES Replaces RFLP infingerprinting technique highly polymorphic high reproducibility identify through absence orpresence of fragment characters can be increased by changing the restriction enzyme and nucleotide at selective primers
- 21. DISADVANTAGES Dominant – lose the codominant character Homology – ability to differentiate different fragment with similar size Mutation rate – high homo plasy - High levels of variation Scoring - bias
- 22. APPLICATIONS Monitoring inheritance of agronomic traits diagnostic in genetically inherited disease pedigree analysis forensic typing (parentage analysis) identifying hybrids species level relationship