Kinases CK1 and GSK3,

sequentially phosphorylate β-catenin at multiple serine and threonine residues

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#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. 

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#Unit 13. Methods in Biology

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

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#Unit 13. Methods in Biology

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

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#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.

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#Unit 13. Methods in Biology

In case of nested PCR, the target sequence is amplified using a pair of PCR primers, and a portion of the amplification product is reamplified using another pair of PCR primers complementary to the regions located immediately beyond the 3'-ends of the first pair of primers.

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#Unit 13. Methods in Biology

In case of nested PCR, the inner primers have a lower annealing temperature than that of the outer pair. For the initial several cycles of PCR the higher denaturation and annealing temperatures are used; as a result, only the outer pair of primer is able to prime amplification.