#Id: 2546

#Unit 13. Methods in Biology

The annealing temperature has a marked influence on PCR efficiency; a temperature higher than the ideal annealing temperature reduces PCR efficiency.

#Id: 2547

#Unit 13. Methods in Biology

Primer sequence also· affects PCR efficiency. For example, if the primers used for PCR have complementary regions, primer dimers will be formed, and PCR efficiency will markedly decline.

#Id: 2548

#Unit 13. Methods in Biology

The sequence of the target DNA itself can affect PCR efficiency. The GC-rich sequences in target DNA may form secondary structures in the single strands produced by denaturation; this could reduce PCR efficiency.

#Id: 2549

#Unit 13. Methods in Biology

7-dcaza guanosine triphosphate (7-deaza OTP) can be used in place of OTP; it reduces hairpin loop formation without affecting the Watson-Crick base pairing required for annealing and primer extension. 

#Id: 2550

#Unit 13. Methods in Biology

Addition of certain proteins, such as, bovine serum albumin enhances efficiency by protecting the DNA polymerase and by binding to PCR inhibitors. 

#Id: 2551

#Unit 13. Methods in Biology

5′ nuclease assay (TaqMan assay). PCR is undertaken with a fluorescence reporter/quencher pair (called RQ probe).