Automated detection of prevalent mutations in BRCA1 and BRCA2 genes, using a fluorogenic PCR allelic discrimination assay

被引:13
作者
Abbaszadegan, MR
Struewing, JP
Brown, KM
Snider, JV
Goodsaid, F
Gore-Langton, R
Hughes, MR
机构
[1] Georgetown Univ, Med Ctr, Inst Mol & Human Genet, Washington, DC 20007 USA
[2] Natl Human Genome Res Inst, NIH, Bethesda, MD 20892 USA
[3] Perkin Elmer Corp, Appl Biosyst Div, Foster City, CA 94404 USA
来源
GENETIC TESTING | 1997年 / 1卷 / 03期
关键词
D O I
10.1089/gte.1997.1.171
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
Mutations in the genes BRCA1 and BRCA2 account for 5%-10% of familial early onset breast cancer. Identification of these mutations allows molecular diagnosis for breast cancer susceptibility. A high through-put automated PCR allelic discrimination assay (ADA) was developed to detect the prevalent mutations in these genes. Two allele specific oligonucleotides (ASO) are directly used in the PCR reaction, in both of which the fluorescent reporter and quencher dyes are attached to the 5' and 3' ends, respectively. During PCR, fluorescence is generated after cleavage of the annealed ASO by the 5' nuclease activity of Tag polymerase. The wild-type BRCA sequence is distinguished from the mutant sequence by the differential fluorescence emission of two different reporter dyes. The sensitivity of ADA is at the level of a single cell following a nested PCR, Eighty-six patient samples can be analyzed for each mutation in 15-min post-PCR without the need for radioactivity, gel electrophoresis, or membrane blotting/hybridization.
引用
收藏
页码:171 / 180
页数:10
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