Primers, in PCR

The Allele-Specific Amplification Assay (ASA) assay is based on the fact that Taq polymerase will not initiate amplification from a primer that has a mismatch at the 3 ends. Two primers are designed so that the 3 base of the primer corresponds to the site of the genetic mutation to be tested, with either the normal or the mutant sequence at the 3 base positions. An unknown sample can then be tested for the presence of the mutation by using both the normal and the mutant primers in PCR with a common reverse primer. If the sample contains only normal sequence, a PCR product will only be produced when the normal primer is used, and similarly when the sample contains mutant sequence a product will only result from use of the mutant primer. Like the PCR-restriction enzyme method discussed, the ASA approach has also been applied to the detection of mutations in the CYP2D6 gene (16). 

Weder JKP, Rehbein H, Kaiser K-P (2001). On the specificity of tuna-directed primers in PCR-SSCP analysis of fish and meat. Eur. Food Res. Technol, 213 139-144. 

Template preparation for pyrosequencing is straightforward. After generation of the template by PCR, the product should be purified prior to pyrosequencing. Unincorporated nucleotides and PCR primers in PCR reaction perturb the pyrosequencing reaction, and the salt in the PCR reaction can inhibit the enzyme system and should be removed or diluted. Depending on the sequence of the DNA template, up to 200 bp can be read. Stretches of three or more identical bases can lead to problems in data interpretation. As for every sequencing reaction, the primer should bind only once at the template. 

The translation of the sequence of the cDNA encoding deacetylvindoline 4-O-acetyltransferase compared to other putative plant acetyltransferases revealed a conserved region near the carboxy terminus of the proteins. This sequence was used to design a degenerate antisense oligodeoxynucleotide primer for PCR. The sense primer was based upon an internal peptide sequence of salutaridinol 7-0-acetyltransferase. This approach finally yielded a partial cDNA that encoded salutaridinol 7-O-acetyltransferase. The full-length clone was obtained by RACE-PCR and was functionally expressed in S. frugiperda Sf9 cells.28 The amino acid sequence of salutaridinol 7-O-acetyltransferase is most similar (37% identity) to that of deacetylvindoline acetyltransferase of C. roseus.27... 

Mix equal amonnts of promoter amplification primers for each of the genes (see Table 1) and for the j3-actin gene. Use 2/lOpL of the mixed primers for each gene in the reaction (for example, if yon are mixing five primer pair sets, use 1 pL of the mixed primers for each reaction, 200 iiM of each primer in the PCR reaction). 

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