Validation Of Snp Array Data

LOH and copy number changes at a specific SNP locus can be validated by direct sequencing of PCR products derived from SNP loci and quantitative PCR using a primer that targets the SNP locus, respectively (49). The DNA sequences of SNP loci can be retrieved from dbSNP databases (21) using the reference SNP identification number The primers that flank the SNP locus can be designed, using the software program Primer3 (57) to generate PCR products of 100-150 base pairs, which covers the SNP locus. The PCR product is sequenced, and the presence of two alleles in the sequencing chromatogram of the blood DNA but only one allele in the sequencing chromatogram of tumor DNA confirms an LOH event at the corresponding SNP locus (Fig. 5). When tissue sections are available, copy number changes can be confirmed by fluorescent in situ hybridization (56,58).

Fig. 5. Validation of SNP loci with LOH by PCR amplification and direct sequencing of both blood and tumor DNA. Both alleles were present in the blood DNA as heterozygous. However, only one allele remained in the tumor DNA.
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