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Polymerase Chain Reaction. What is PCR History of PCR How PCR works Optimizing PCR Fidelity, errors & cloning PCR primer design Application of PCR. Adaptor Ligation. Two different adaptors (short double stranded DNA sequences with sticky end) are ligated to the digested fragments.
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Polymerase Chain Reaction What is PCR History of PCR How PCR works Optimizing PCR Fidelity, errors & cloning PCR primer design Application of PCR
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.
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.
Electrophoresis • polyacrylamide gel is used for separating DNA bands. • Normally, 30-100 DNA bands can be detected by AFLP on polycrylamide gel.
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.
