Using a Single-Capillary Bidirectional Sequencing for Genotyping the TGFβ1 Gene
a Institute of Clinical Biochemistry and Diagnostics, b Department of Oncology and Radiotherapy, Faculty of Medicine and Faculty Hospital, Hradec Králové
A novel single-capillary method of verification of both-strand analysis of the -1552delAGG genetic polymorphism in the TGF β1 gene is presented. For the verification we used the single-tube sequencing reaction with forward and reverse primers modified at the 5'-end by adap¬ters composed of 55 (forward primer) and 10 (reverse primer) repetitive units of cytosine and adenine (CA adap¬ters). The CA adapters made it possible to get complete readings (of both strands of the amplicons) and to determine different migration times for forward and reverse sequencing fragments in single-capillary electrophoresis. The frequencies of the -1552delAGG variant in experimental (46 women) and control (102 women) groups were 29 % and 33 %, respectively. Our study showed that the attachment of the CA adapters to the primers made it possible to perform sequencing of both strands in one PCR tube as well as to separate forward and reverse sequencing fragments in one capillary.
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