Ligase chain reaction coupled with rolling circle amplification for high sensitivity detection of single nucleotide polymorphisms

Abstract

We present a highly sensitive and homogeneous assay for the detection of single nucleotide polymorphisms (SNPs) by ligase chain reaction (LCR) coupled with rolling circle amplification (RCA). The LCR probes include one pair of probes and a padlock probe (PLP). In the LCR, one pair of probes composed of X and Y, perfectly hybridize with the upper strand of the target DNA after thermal denaturation. They are then ligated by the thermostable ligase to form the ligation product of XY. At the same time, the PLP hybridizes with the lower strand of the target DNA and are ligated to form the circular PLP (cPLP). After repeated cycles of denaturation, annealing, and ligation, the target DNA is amplified exponentially to generate a large number of XY and cPLPs. Subsequently, RCA is triggered by the cPLP as a template and XY as a primer, producing large numbers of long strand DNA products, which are detected by binding with the fluorescent dye, SYBR Green I, in a homogeneous manner. This method is simple, and avoids the need for detection of the LCR products with labeled probes and complex separation steps. The assay is sensitive and specific enough to detect a 1 fM target DNA molecule. It is possible to accurately determine the allele frequency as low as 1.0%. The LCR coupled with RCA assay extends the application of the LCR and RCA, and provides a new strategy for detecting SNPs as well as nucleic acid analysis, immunoassay, and molecular diagnosis.