ThrRS has the opposite problem: It must synthesize Thr–tRNAThr but not Val–tRNAThr. Specificity is conferred by the aminoacylation site, which contains a Zn2+ ion that is coordinated by the side chain OH group of threonine. Valine cannot coordinate the Zn2+ in this way and hence does not undergo adenylylation by ThrRS.  A separate editing site deals with misacylated Ser–tRNAThr. TyrRS distinguishes between tyrosine and phenylalanine through hydrogen missing from the repertoire are GlnRS and asparaginyl-tRNA synthetase (AsnRS). To synthesize Gln-tRNAGln and Asn-tRNAAsn, these organisms possess distinct glutamyl-tRNA synthetase (GluRS) and aspartyl-tRNA synthetase (AspRS) enzymes that are nondiscriminating (ND). GluRSND synthesizes both Glu-tRNAGlu as well as misacylated Glu-tRNAGln; AspRSND synthesizes both Asp-tRNAAsp and misacylated Asp-tRNAAsn.

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#Unit 13. Methods in Biology

Touchdown PCR, has been devised to increase the specificity of PCR without lowering the efficiency.

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#Unit 13. Methods in Biology

Touchdown  PCR is initiated at very high annealing temperatures, which allow only perfectly matched primer-template DNA hybrids to form and support amplification. The annealing temperature is dropped in a stepwise fashion with each cycle of PCR. 

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#Unit 13. Methods in Biology

The logic for hotstart PCR is as follows, most DNA samples used as template will have some single-stranded DNA. 

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#Unit 13. Methods in Biology

In case of hotstart PCR, After the reaction mixture is prepared, primers can pair to the single-stranded regions in a nonspecific manner. 

A more attractive alternative is to cover the reaction mixture with a plug of inert wax and place the critical reagent on top of this plug. 

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#Unit 13. Methods in Biology

A more attractive alternative is to cover the reaction mixture with a plug of inert wax and place the critical reagent on top of this plug. 

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#Unit 13. Methods in Biology

Oligonucleotide-directed mutagenesis

The basic PCR mutagenesis system involves the use of two primary PCR reactions to produce two overlapping DNA fragments, both bearing the same mutation in the overlap region; this technique is thus termed overlap extension PCR .