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Sequencing of simple sequence repeat anchored polymerase chain reaction amplification products of Biomphalaria glabrata

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Author(s): Caldeira Roberta L | Carvalho Omar S | Lage Regina CG | Cardoso Paula Cristina M | Oliveira Guilherme C

Journal: Memórias do Instituto Oswaldo Cruz
ISSN 0074-0276

Volume: 97;
Issue: suppl.1;
Start page: 23;
Date: 2002;
Original page

Keywords: Biomphalaria glabrata | microsatellites | simple sequence repeat | polymerase chain reaction

ABSTRACT
Simple sequence repeat anchored polymerase chain reaction amplification (SSR-PCR) is a genetic typing technique based on primers anchored at the 5' or 3' ends of microsatellites, at high primer annealing temperatures. This technique has already been used in studies of genetic variability of several organisms, using different primer designs. In order to conduct a detailed study of the SSR-PCR genomic targets, we cloned and sequenced 20 unique amplification products of two commonly used primers, CAA(CT)6 and (CA)8RY, using Biomphalaria glabrata genomic DNA as template. The sequences obtained were novel B. glabrata genomic sequences. It was observed that 15 clones contained microsatellites between priming sites. Out of 40 clones, seven contained complex sequence repetitions. One of the repeats that appeared in six of the amplified fragments generated a single band in Southern analysis, indicating that the sequence was not widespread in the genome. Most of the annealing sites for the CAA(CT)6 primer contained only the six repeats found within the primer sequence. In conclusion, SSR-PCR is a useful genotyping technique. However, the premise of the SSR-PCR technique, verified with the CAA(CT)6 primer, could not be supported since the amplification products did not result necessarily from microsatellite loci amplification.
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