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Polymerase Chain Reaction

Polymerase Chain Reaction. What is PCR History of PCR How PCR works Optimizing PCR Fidelity, errors & cloning PCR primer design Application of PCR. AFLP Amplified Fragment Length Polymorphism. AFLP. - a method based on PCR developed in 1995 by Vos et al.

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Polymerase Chain Reaction

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  1. Polymerase Chain Reaction What is PCR History of PCR How PCR works Optimizing PCR Fidelity, errors & cloning PCR primer design Application of PCR

  2. AFLPAmplified Fragment Length Polymorphism

  3. AFLP • - a method based on PCR developed in 1995 by Vos et al. • Involves the use of RFLP and PCR techniques • Compared with the widely used RFLP, AFLP is faster, less labour intensive and provide more information. • An additional advantage over RAPD is their reproducibility.

  4. AFLP 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.

  5. AFLP Procedures in AFLP: - Digestion - Adaptor Ligation - Amplification - Electrophoresis

  6. STEP 1: Restriction-Ligation

  7. STEP 2: Pre-selective PCR EcoRI PRE-SELECTIVE PRIMER GTAGACTGCGTACC AATT CA CA AT GAGTCCTGAGTA MseI PRE-SELECTIVE PRIMER

  8. GTAGACTGCGTACC AATT CACT STEP 3: Selective PCR SELECTIVE PRIMER FAM EcoRI SELECTIVE PRIMER (labeled) GTAGACTGCGTACC AATT CA CA AT GAGTCCTGAGTA GACA AT GAGTCCTGAGTA MseI SELECTIVE PRIMER

  9. MseI EcoRI MseI MseI MseI MseI MseI MseI MseI MseI MseI MseI MseI MseI MseI MseI EcoRI EcoRI EcoRI MseI MseI EcoRI MseI MseI MseI MseI MseI MseI MseI MseI MseI MseI EcoRI MseI MseI MseI MseI MseI MseI MseI MseI MseI MseI MseI EcoRI: 6bp cutter --> one cut every 4096 bp MseI: 4bp cutter --> one cut every 256 bp Selective PCR product contains many unlabeled fragments that will not be visible on ABI

  10. Number of bands in AFLP profile is determined by 1 Genome size: larger genome ---> more bands 2 Number of selective nucleotides in selective primers 3 Dilution of PCR product Low (noise) peaks get magnified Why optimize number of bands? 1 Size homoplasy !!!!! 2 Difficult to score

  11. Reproducibility High reproducibility has generally been reported However, DNA quality is crucial component (use same DNA extraction protocol for all samples!) Assess quality of data by repeating several samples from scratch i.e. starting with DNA extraction

  12. Optimizing AFLP reactions 1 DNA quality 2 DNA quality A successful AFLP analyses depends crucially on this 3 DNA quality 4 Increase restriction time to 2 hours 5 Increase ligation time to 16 hours 6 Use fresh T4 ligase 7 Increase amount of DNA (rest-lig) added to pre-selective PCR (15 ul DNA’ in 50ul reaction) 8 Reduce amount of DNA in Selective PCR 9 Increase amount of cycles in Selective PCR 10 Increase amount of TAQ in Selective PCR 11 Several people have reported better results with TaqI vs MseI (but this requires different adaptors)

  13. Digestion Two different restriction endonucleases are used in digestion. One is 4-base cutter (MseI) and the other one is 6-base cutter (EcoRI). MseI 5’TTAA3’ EcoRI 5’GAATTC3’

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