Murray V, Monchawin C, Cairns M J, England P R, Leigh D, McDonald B L
School of Biochemistry and Molecular Genetics, University of New South Wales, Kensington, Australia.
Hum Mutat. 1993;2(2):118-22. doi: 10.1002/humu.1380020210.
A method is described for the detection of restriction fragment length polymorphisms (RFLPs) in single copy genes in mammalian cells using one 5'-labelled oligonucleotide. This linear amplification (LA) method employs a single oligonucleotide as primer, which is extended by Taq DNA polymerase up to a restriction enzyme cleavage site. The products are arithmetically amplified by thermal cycling. The size of the products are determined by the sequence of the oligonucleotide and the position of the restriction enzyme cleavage site. Hence, an RFLP can be observed by measuring the size of the products. Polymorphisms which differ in size by a small number of base pairs, as are found in (CA)n repeats, are especially suitable for analysis by the LA procedure since the products are run on DNA sequencing gels. A number of genes were examined by the procedure and all produced a satisfactory signal including GC-rich template. It is proposed that the LA method would be suitable for large-scale genetic linkage analysis. The LA procedure has many advantages including the ability to multiplex signals under the same conditions, and lower cost since only one primer is needed.
描述了一种使用一个5'端标记的寡核苷酸来检测哺乳动物细胞单拷贝基因中限制性片段长度多态性（RFLP）的方法。这种线性扩增（LA）方法采用单个寡核苷酸作为引物，由Taq DNA聚合酶延伸至限制性内切酶切割位点。产物通过热循环进行算术扩增。产物的大小由寡核苷酸的序列和限制性内切酶切割位点的位置决定。因此，通过测量产物的大小可以观察到RFLP。在（CA）n重复序列中发现的大小相差少数碱基对的多态性，特别适合用LA程序进行分析，因为产物在DNA测序凝胶上进行电泳。通过该程序检测了多个基因，所有基因都产生了令人满意的信号，包括富含GC的模板。有人提出LA方法适用于大规模遗传连锁分析。LA程序有许多优点，包括能够在相同条件下对信号进行多重分析，并且由于只需要一个引物，成本较低。
