Primers mismatches

Total RNA is isolated from the lymphocytes according to standard procedures and used as a template for radioactive labeled cDNA synthesis. The purified cDNA is used as probe for cDNA expression arrays. The advantages of this method as compared to other array systems are as follows (1) Radioactive-labeled probes are more sensitive than fluorescent-labeled probes and therefore need less sample RNA. (2) The primers used in the cDNA synthesis match the genes represented on the array. (3) The primer sequences are longer compared to other array systems, which increases the hybridization fidelity of RNA to the matching correct set of genes and therefore reduces mismatch reactions. 

Davidow LS (1992) Selecting PCR designed mismatch primers to create diagnostic restriction sites. Comput Appl Biosci 8 193-194... 

Figure 14.8 The original scheme for oligodeoxynucleotide-directed mutagenesis. The mismatched primer is designed to mutate the codon for cysteine (TGC) to that for serine (AGC).

Illustration of a general method of mutagenesis using PCR. Primers are represented as short lines with arrowheads pointing in the 3 direction. The bump in primers 2 and 3 and their products represent a mismatched base, a deliberate alteration in base sequence from that present in the starting DNA. Of the four major products resulting from step 3 only D is extendable by DNA polymerase. 

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). 

The 3 -+5 exonuclease activity plays an important role in polymerization in proof reading the base pair formed at each polymerization step. The enzyme checks the nature of each base-paired primer terminus before the polymerase proceeds to add the next nucleotide to the primer. It thus supplements the capacity of the polymerase to match the incoming nucleotide substrate to the template. A mismatched terminal nucleotide on the primer activates a site on the enzyme which results in the hydrolysis of the phosphodiester bond and the removal of the mismatched residue. The function of this 3 - 5 exonuclease activity is therefore to recognize and cleave incorrectly or non-base paired residues at the 3 -end of DNA chains. It will therefore degrade single stranded DNA and frayed or non-base paired residues at the ends of duplex DNA molecules provided they terminate in a 3 -hydroxyl group. 

There are several possible solutions to the expansion mismatch problem. One is to use a resilient adhesive that deforms with the substrate during temperature change. The penalty in this case is possible creep of the adhesives, and highly deformable adhesives usually have low cohesive strength. Another approach is to adjust the thermal expansion coefficient of the adhesive to a value that is nearer to that of the substrate. This is generally accomplished by selection of a different adhesive or by formulating the adhesive with specific fillers to tailor the thermal expansion. A third possible solution is to coat one or both substrates with a primer. This substance can provide either resiliency at the interface or an intermediate thermal expansion coefficient that will help reduce the overall stress in the joint. 

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