Walker G T, Fraiser M S, Schram J L, Little M C, Nadeau J G, Malinowski D P
Department of Molecular Biology, Becton Dickinson Research Center, Research Triangle Park, NC 27709.
Nucleic Acids Res. 1992 Apr 11;20(7):1691-6. doi: 10.1093/nar/20.7.1691.
Strand Displacement Amplification (SDA) is an isothermal, in vitro nucleic acid amplification technique based upon the ability of HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo- klenow) to extend the 3'-end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as target for an antisense reaction and vice versa. In the original design (G. T. Walker, M. C. Little, J. G. Nadeau and D. D. Shank (1992) Proc. Natl. Acad. Sci 89, 392-396), the target DNA sample is first cleaved with a restriction enzyme(s) in order to generate a double-stranded target fragment with defined 5'- and 3'-ends that can then undergo SDA. Although effective, target generation by restriction enzyme cleavage presents a number of practical limitations. We report a new target generation scheme that eliminates the requirement for restriction enzyme cleavage of the target sample prior to amplification. The method exploits the strand displacement activity of exo- klenow to generate target DNA copies with defined 5'- and 3'-ends. The new target generation process occurs at a single temperature (after initial heat denaturation of the double-stranded DNA). The target copies generated by this process are then amplified directly by SDA. The new protocol improves overall amplification efficiency. Amplification efficiency is also enhanced by improved reaction conditions that reduce nonspecific binding of SDA primers. Greater than 10(7)-fold amplification of a genomic sequence from Mycobacterium tuberculosis is achieved in 2 hours at 37 degrees C even in the presence of as much as 10 micrograms of human DNA per 50 microL reaction. The new target generation scheme can also be applied to techniques separate from SDA as a means of conveniently producing double-stranded fragments with 5'- and 3'-sequences modified as desired.
链置换扩增(SDA)是一种等温体外核酸扩增技术,其基于HincII切割其识别位点的半硫代磷酸酯形式的未修饰链的能力,以及核酸外切酶缺陷型klenow(exo-klenow)在切口处延伸3'端并置换下游DNA链的能力。指数扩增源于将正向和反向反应偶联,其中从正向反应中置换出的链作为反向反应的靶标,反之亦然。在最初的设计中(G.T.沃克、M.C.利特尔、J.G.纳多和D.D.尚克(1992年)《美国国家科学院院刊》89,392 - 396),首先用限制性内切酶切割靶DNA样品,以产生具有确定的5'和3'末端的双链靶片段,然后该片段可进行SDA。尽管有效,但通过限制性内切酶切割产生靶标存在一些实际限制。我们报告了一种新的靶标生成方案,该方案消除了扩增前对靶样品进行限制性内切酶切割的要求。该方法利用exo-klenow的链置换活性来生成具有确定的5'和3'末端的靶DNA拷贝。新的靶标生成过程在单一温度下发生(双链DNA初始热变性后)。通过该过程生成的靶拷贝然后直接通过SDA进行扩增。新方案提高了整体扩增效率。通过改善反应条件减少SDA引物的非特异性结合,扩增效率也得到了提高。即使在每50微升反应中存在多达10微克人DNA的情况下,在37℃下2小时内也能实现结核分枝杆菌基因组序列大于10^7倍的扩增。新的靶标生成方案也可应用于与SDA分开的技术,作为方便地产生具有按需要修饰的5'和3'序列的双链片段的一种手段。