Single Nucleotide Primer Extension for Analysis of Sequence Variants
In the DNA single nucleotide primer extension (SNuPE) assay for detection of known mutations (Kuppuswamy et al., 1991), the DNA segment containing the mutation is amplified by PCR and the product is isolated (Fig. 25.1). Then primer extension is done on aliquots of the PCR fragment with the SNuPE primer that ends just one nucleotide 5′ to the base difference. In this step only one radioactive nucleotide is used per aliquot, the one corresponding to the wild-type (Wt) sequence in one tube, and the one corresponding to the mutant sequence in the other. In one tube the radionucleotide corresponding to the Wt sequence can extend the primer on the Wt template but cannot extend the primer on the mutant fragment. In the other tube the opposite is the case. On a fragment amplified from an individual heterozygous for the given point mutation, each radionucleotide incorporates in the separate SNuPE reactions. The SNuPE primers are separated from unincorporated nucleotides by denaturing polyacrylamide gel electrophoresis and the results are visualized by autoradiography. The PCR amplification step before the primer extension makes this assay very sensitive. If the original DNA was a mix of mutant and Wt DNA, both nucleotides incorporate on a mixed template, and this enables one to screen pooled samples for mutation as performed by Krook et al. (1992). These authors also described a more economical variation of the method, called “multiplex SNuPE,” that allows simultaneus examination of multiple loci in the same reaction and on the same gel. They amplified three different fragments in the same PCR reaction and did the SNuPE assay with three different SNuPE primers which differed in length by five nucleotides.
KeywordsBase Difference Mixed Template Mutant Fragment Fragment Isolation Single Nucleotide Prime Extension
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