Jednosměrné sekvenování PCR produktů MBL2 genu pomocí polyadenylovaného sekvenačního primeru

Abstrakt

Standard dideoxynucleotide sequencing is not able to provide complete sequences of analysed amplicons. First 25–50 nucleotides after the 3' end of sequencing primers are usually lost because of cleaning procedures performed to remove excess of primers, nucleotides, salts, and other contaminants. A new possibility is described of increasing readability of the initial part of short PCR amplicons using a sequencing primer modified by a polyadenylated tail attached to its 5' end. 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. The examined sites lie near both ends of the products. Purified amplicons were used in cycle sequencing reactions containing either conventional or polyadenylated forward primers. The data obtained by the former approach did not contain the initial 25-nucleotide segment with 5' UTR, and double-strand sequencing was necessary. In contrast, the polyadenylated primers enabled complete and reliable readability of the amplicons. Both the approaches provided the same results. 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. A broader study is needed to confirm our preliminary results. We revealed that the polyadenylated primer used in the cycle sequencing extends the readable range of MBL2 amplicons without necessity of double-strand sequencing, subcloning or reamplification with other pair of primers.