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Single-Strand Sequencing of PCR Products of the MBL2 Gene with a Polyadenylated Sequencing Primer

Publication at Faculty of Medicine in Hradec Králové |
2012

Abstract

In a group of 24 patients with recurrent vulvovaginal candidiasis and 24 healthy subjects we analysed polymorphic sites in the 5' untranslated region (5' UTR) and codons 52, 54, and 57 of the MBL2 gene. Using PCR we amplified 317 bp MBL2 products covering all the polymorphisms.

Purified amplicons were used in cycle sequencing reactions containing conventional or polyadenylated forward primers. We revealed that the polyadenylated primer used in the cycle sequencing extends the readable range of MBL2 amplicons without necessity of doublestrand sequencing, subcloning or reamplification with other pair of primers.

Our preliminary data showed that the distribution of MBL2 alleles, haplotypes, and genotypes in the patients and healthy subjects was very similar and the differences were not statistically significant.