Sequencing of simple sequence repeat anchored polymerase chain reaction amplification products of Biomphalaria glabrata.
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.
Caldeira
Roberta L
RL
Laboratório de Helmintoses Intestinais, Centro de Pesquisas René Rachou, Fiocruz, Belo Horizonte, MG, 30190-002, Brasil.
Carvalho
Omar S
OS
Lage
Regina C G
RC
Cardoso
Paula Cristina M
PC
Oliveira
Guilherme C
GC
D000818:Animals; D001483:Base Sequence; D001700:Biomphalaria / Q000235:genetics*; D015139:Blotting, Southern; D001938:Brazil; D004247:DNA / Q000032:analysis; D007303:Insect Vectors / Q000235:genetics*; D018598:Minisatellite Repeats / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016622:Silver Staining
D004247:DNA