Amplified Fragment length Polymorphism

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Slide 1: 

…….AGCTAATCTAGAGAGAGAGAGAGCTAGCTGATTCAACTAC……. …….TCGATTAGATCTCTCTCTCTCTCGATCGACTATGTTGATG……. New strand New strand AFLP GATTAGATCTCTCTC Long Fixed Primer (about 15 bp) CTA Short Random Primer (2-4 bp) No cloning required No sequencing required Long Fixed Preimer + a small random primer is used Detects several loci simultaneously > RAPD > RFLP Dominant marker Useful for fingerprinting and phylogenetic studies Long primer has a fixed position, so is reproducible Not useful for comparative mapping Requires more facilities and skill

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Mapping of Rf1 gene in sorghum AFLP Combination of RFLP and RAPD analysis AFLP combines the specificity of restriction analysis with PCR amplification The sequence variation detected is the same as that detected by RFLP analysis, but the number of polymorphisms detected per analysis is higher AFLP uses restriction enzyme-digested genomic DNA as the template for a PCR reaction with primers that contain the restriction enzyme recognition site as well as additional ‘arbitrary’ nucleotides that extend beyond the restriction site. By varying the number of these additional ‘arbitrary’ nucleotides that extend beyond the restriction sites into the unknown sequence, it is possible to control the proportion of the ligated fragments that could be amplified. The amplified products are then resolved by polyacrylamide gel electrophoresis. In general, 75 to 150 fragments are amplified with each primer combination, and as each fragment represents a unique site, the proportion of the genome assayed with each primer combination is much higher than with any other DNA analysis approach. 15-20 minutes

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Sterile Fertile Bulk Sterile Bulk Fertile LICOR GEL Total No. of bands = 130-150

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