Abravaya K, Carrino J J, Muldoon S, Lee H H
Abbott Laboratories, Probe Diagnostics Business Unit, Abbott Park, IL 60064.
Nucleic Acids Res. 1995 Feb 25;23(4):675-82. doi: 10.1093/nar/23.4.675.
DNA amplification systems are powerful technologies with the potential to impact a wide range of diagnostic applications. In this study we explored the feasibility and limitations of a modified ligase chain reaction (Gap-LCR) in detection and discrimination of DNAs that differ by a single base. LCR is a DNA amplification technology based on the ligation of two pairs of synthetic oligonucleotides which hybridize at adjacent positions to complementary strands of a target DNA. Multiple rounds of denaturation, annealing and ligation with a thermostable ligase result in the exponential amplification of the target DNA. A modification of LCR, Gap-LCR was developed to reduce the background generated by target-independent, blunt-end ligation. In Gap-LCR, DNA polymerase fills in a gap between annealed probes which are subsequently joined by DNA ligase. We have designed synthetic DNA targets with single base pair differences and analyzed them in a system where three common probes plus an allele-specific probe were used. A single base mismatch either at the ultimate 3' end or penultimate 3' end of the allele specific probe was sufficient for discrimination, though better discrimination was obtained with a mismatch at the penultimate 3' position. Comparison of Gap-LCR to allele-specific PCR (ASPCR) suggested that Gap-LCR has the advantage of having the additive effect of polymerase and ligase on specificity. As a model system, Gap-LCR was tested on a mutation in the reverse transcriptase gene of HIV, specifically, one of the mutations that confers AZT resistance. Mutant DNA could be detected and discriminated in the presence of up to 10,000-fold excess of wild-type DNA.
DNA扩增系统是强大的技术，有可能影响广泛的诊断应用。在本研究中，我们探讨了改良连接酶链反应（间隙连接酶链反应，Gap-LCR）在检测和区分相差单个碱基的DNA方面的可行性和局限性。连接酶链反应（LCR）是一种基于两对合成寡核苷酸连接的DNA扩增技术，这两对寡核苷酸在相邻位置与目标DNA的互补链杂交。通过热稳定连接酶进行多轮变性、退火和连接，导致目标DNA呈指数扩增。为了减少非靶标依赖性平端连接产生的背景，开发了一种LCR的改良方法——间隙连接酶链反应（Gap-LCR）。在Gap-LCR中，DNA聚合酶填补退火探针之间的间隙，随后由DNA连接酶连接。我们设计了具有单个碱基对差异的合成DNA靶标，并在一个使用三种通用探针加一个等位基因特异性探针的系统中对其进行分析。等位基因特异性探针的最终3'端或倒数第二个3'端的单个碱基错配足以进行区分，不过在倒数第二个3'位置出现错配时区分效果更好。将Gap-LCR与等位基因特异性PCR（ASPCR）进行比较表明，Gap-LCR具有聚合酶和连接酶对特异性的累加效应这一优势。作为一个模型系统，在HIV逆转录酶基因的一个突变上测试了Gap-LCR，具体来说，是赋予齐多夫定（AZT）抗性的突变之一。在存在高达10000倍过量野生型DNA的情况下，能够检测和区分突变DNA。
