TLS Online TPP Program

#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.

TLS Online TPP Program

#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. 

TLS Online TPP Program

#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. 

TLS Online TPP Program

#Id: 2551

#Unit 13. Methods in Biology

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

TLS Online TPP Program

#Id: 2552

#Unit 13. Methods in Biology

During extension by Taq polymerase, the probe is cleaved owing to the nuclease activity of the polymerase.

TLS Online TPP Program

#Id: 2553

#Unit 13. Methods in Biology

Due to release of the reporter, there is a detectable increase in fluorescence, which is monitored in real-time PCR experiments.