Automated detection of trinucleotide repeats in fragile X syndrome

被引:20
作者
Hamdan, H
Tynan, JA
Fenwick, RA
Leon, JA
机构
[1] Nichols Inst, Quest Diagnost, Dept Biotechnol Res & Dev, San Juan Capistrano, CA 92690 USA
[2] Nichols Inst, Quest Diagnost, Dept Mol Biol, San Juan Capistrano, CA 92690 USA
来源
MOLECULAR DIAGNOSIS | 1997年 / 2卷 / 04期
关键词
genetics; polymerase chain reaction; fluorescence; betaine;
D O I
10.1016/S1084-8592(97)80037-9
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Background: The conventional method for diagnosis of fragile X syndrome has been amplification of the trinucleotide repeat region of the FIMR-1 gene by polymerase chain reaction (PCR) and Southern blot analysis to detect full expansion and hypermethylation. "Stuttering" resulting from incomplete amplification is still observed in the PCR products despite the use of reagents that reduce the secondary structure of the GC-rich template. In addition, PCR products can be detected by autoradiography only after 1 to 2 days of exposure. By combination of a recently reported amplification protocol with fluorescence detection of PCR products in an automated DNA sequencer, the PCR protocol for amplification of trinucleotide repeats was simplified. This modified protocol is highly reproducible, more accurate, and less costly than the conventional protocol because of the elimination of radioisotopes from the PCR. Methods and Results: PCRs were conducted with betaine and Pfu DNA polymerase. This improved PCR protocol allowed immediate detection of PCR products in agarose gels containing ethidium bromide, Stuttering was completely eliminated and fragments of up to 1 kb (similar to 250 repeats) were visible in agarose gels. PCR products were automatically detected by laser fluorescence in an automated DNA sequencer by inclusion of a fluorescently-labeled primer in the PCR reaction. A short electrophoresis run of 100 minutes in denaturing acrylamide gels was sufficient to give high resolution of fragments with higher accuracy and sensitivity than conventional detection by autoradiography. Conclusions: A simple, nonradioactive protocol that is more rapid and less expensive than the conventional PCR protocol for the detection of trinucleotide repeats has been developed. By use of this detection protocol, fragment sizes containing up to 100 repeats could be detected, alleles differing by one trinucleotide repeat were clearly resolved, and heterogeneous repeat patterns such as those present in mosaics could be discriminated. This protocol has been adapted to the amplification and detection of at least two other classes of trinucleotide repeats [(CAG)(n) and (CTG)(n)], suggesting that it may be a universal protocol for PCR amplification and detection of trinucleotide repeats.
引用
收藏
页码:259 / 269
页数:11
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