Wehnert M S, Matson R S, Rampal J B, Coassin P J, Caskey C T
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030.
Nucleic Acids Res. 1994 May 11;22(9):1701-4. doi: 10.1093/nar/22.9.1701.
Oligonucleotides representing 60 trinucleotide (21mers) and four dinucleotide (20mers) tandem repeats were directly synthesized and arrayed onto an aminated polypropylene substrate. DNA samples of different complexities (a CAG-containing 21mer oligonucleotide, PCR fragments of 200 to 3,000 bp, and cosmids with 31 to 35 kb inserts) were radiolabelled and hybridized to the oligonucleotide array at various temperatures. When compared to sequence data available from the test DNAs, the reverse blot system specifically identified various tri- and dinucleotide short tandem repeats (STRs) in every case. Moreover, there was no random or cross hybridization to nonspecific sequences. It was possible to detect as few as three repeated units in a particular location, as shown for (CCT)n, (GCC)n and (CAC)n triplets in cosmid DNA. Varying the hybridization stringency can enhance the detection of STRs. This single-step reverse blot system therefore allows the rapid, specific and sensitive identification of various STRs in DNA sources of different complexity.
代表60个三核苷酸（21聚体）和4个二核苷酸（20聚体）串联重复序列的寡核苷酸被直接合成并排列在胺化聚丙烯基质上。不同复杂度的DNA样本（含CAG的21聚体寡核苷酸、200至3000 bp的PCR片段以及插入片段为31至35 kb的黏粒）经放射性标记后，在不同温度下与寡核苷酸阵列杂交。与测试DNA的可用序列数据相比，该反向杂交系统在每种情况下都能特异性识别各种三核苷酸和二核苷酸短串联重复序列（STR）。此外，不存在与非特异性序列的随机或交叉杂交。如在黏粒DNA中对（CCT）n、（GCC）n和（CAC）n三联体所示，在特定位置可以检测到少至三个重复单元。改变杂交严谨度可增强对STR的检测。因此，这种单步反向杂交系统能够快速、特异性且灵敏地识别不同复杂度DNA来源中的各种STR。
