Marshall R L, Laffler T G, Cerney M B, Sustachek J C, Kratochvil J D, Morgan R L
Abbott Laboratories, Molecular Diagnostics, Abott Park, Illinois 60064, USA.
PCR Methods Appl. 1994 Oct;4(2):80-4. doi: 10.1101/gr.4.2.80.
The ligase chain reaction (LCR) and the gap ligase chain reaction (gLCR) are exponential amplification techniques for the detection of DNA sequences in a sample. Both techniques depend on the enzyme, DNA ligase, to join adjacent probes annealed to a DNA molecule. However, DNA ligase joins DNA inefficiency on an RNA target. Consequently, LCR and gLCR cannot amplify RNA efficiency. RNA detection methods using LCR or gLCR require a cDNA synthesis step. The carryover of four dNTPs from the cDNA reaction inhibits gLCR. Although LCR can use cDNA reaction products directly, background generated by blunt-end ligation does not allow the high sensitivity typically needed for HIV or HCV detection. The asymmetric gap ligase chain reaction (AGLCR) is a modification of gLCR that allows for the detection of RNA by using < or = 3 of the 4 nucleotides in the cDNA step and the gLCR step. Fewer than 50 copies of synthetic RNA transcript can be reproducibly detected. HCV, an RNA virus with no DNA intermediate, was chosen as the initial RNA model system. HCV antibody-positive and normal samples were analyzed, and the results were found to correlate with the results obtained using nested RNA-PCR. AGLCR provides a new nucleic acid amplification technique that can aid in the diagnosis of disease when the detection of RNA is critical.
连接酶链式反应（LCR）和缺口连接酶链式反应（gLCR）是用于检测样本中DNA序列的指数扩增技术。这两种技术都依赖于DNA连接酶将与DNA分子退火的相邻探针连接起来。然而，DNA连接酶对RNA靶标的连接效率较低。因此，LCR和gLCR无法有效地扩增RNA。使用LCR或gLCR的RNA检测方法需要一个cDNA合成步骤。cDNA反应中四种dNTP的残留会抑制gLCR。虽然LCR可以直接使用cDNA反应产物，但平端连接产生的背景信号无法满足HIV或HCV检测通常所需的高灵敏度。不对称缺口连接酶链式反应（AGLCR）是gLCR的一种改进方法，它通过在cDNA步骤和gLCR步骤中使用4种核苷酸中的≤3种来实现对RNA的检测。可重复检测到少于50个拷贝的合成RNA转录本。HCV是一种没有DNA中间体的RNA病毒，被选作初始的RNA模型系统。对HCV抗体阳性和正常样本进行了分析，结果发现与使用巢式RNA-PCR获得的结果相关。当RNA检测至关重要时，AGLCR提供了一种新的核酸扩增技术，有助于疾病的诊断。
