Genomic DNA was extracted from peripheral blood lymphocytes of the donated blood samples. Amplimers were designed to cover the coding region and intron–exon boundaries of the two published exons (NM_001029883.1). Primers were designed to avoid the known polymorphisms, and their properties were evaluated with OligoCalc
8 (primer sequences and PCR conditions are available on request). The resulting amplimers ranged from 322 to 598 base pairs. Two methods of variation screening were used. On the basis of the reported polymorphisms in
C2ORF71 at the time of the experiment design (18 polymorphic sites, NCBI dbSNP database, November 2009), the amplimers were divided in two groups:
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Four of 10 (group 1), covering two or more polymorphic sites each, were amplified by PCR, and mutation analysis was performed by direct sequencing of all PCR products.
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Six of 10 amplimers (group 2), covering one or no polymorphic sites each, were analyzed by PCR (with highly saturating fluorescent dye), HRM analysis, and Sanger sequencing.
An in-scale schematic of the
C2ORF71 coding region, showing the coverage by either HRM or direct sequencing and the polymorphism distribution before and after the study, is presented in
Table 1.
The decision to perform direct sequencing on group 1 and HRM on group 2 was based on the assumption that three or more common polymorphic sites in an amplimer would impede the analysis of HRM curves (for example, small differences in fluorescence would not be highlighted).
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