Ligase detection reaction for identification of low abundance mutations

Direct Detection of Mutant DNA in a Mixed Population of Higher Copy Number Wild-Type DNA Based on Ligase Detection Reaction in Conjunction with Fluorescence Resonance Energy Transfer

“…[2][3][4][5][6][7] In the PCR/LDR assays, following PCR amplification of the appropriate gene fragments, which contain sections of the gene(s) that possess the point mutations, the amplicon is mixed with two LDR primers (common primer and discriminating primer) that flank the mutation of interest at an appropriate temperature. The discriminating primer contains a base at its 3′-end that coincides with the single base mutation site.…”

“…If there is a mismatch, no ligation of the two primers will occur. On the other hand, a perfect match results in a successful ligation of the two primers and produces a product that can be analyzed in a variety of fashions such as gel electrophoresis 3,4,7 and hybridization. 2,5,6 However, those post-LDR processes to separate the LDR products from the residual primers prior to detection are cumbersome, are the most time-consuming step in the entire assay procedure, making it difficult to quickly determine if mutated DNAs are present in samples.…”

A Fluorescence Quenching Assay Based on Molecular Beacon Formation through a Ligase Detection Reaction for Facile and Rapid Detection of Point Mutations

“…4 The ligase detection reaction (LDR) is a useful technique that allows for discriminative detection of rare mutations present within a sample containing primarily wildtype DNA. 5 In the LDR assay, a successful ligation between two LDR primer ends (a discriminating primer and a common primer) occurs in the presence of DNA ligase only if these primers are perfectly complementary to the targeted DNA, especially at the 3′-end of the discriminating primer. Selective detection of the LDR products (i.e., the ligated fragments) to determine the presence of a mutation has conventionally been performed by either electrophoretic sorting [5][6][7][8][9] or DNA-DNA hybridization.…”

“…5 In the LDR assay, a successful ligation between two LDR primer ends (a discriminating primer and a common primer) occurs in the presence of DNA ligase only if these primers are perfectly complementary to the targeted DNA, especially at the 3′-end of the discriminating primer. Selective detection of the LDR products (i.e., the ligated fragments) to determine the presence of a mutation has conventionally been performed by either electrophoretic sorting [5][6][7][8][9] or DNA-DNA hybridization. [10][11][12][13][14] However, the procedures involved in these techniques are generally labor-intensive and time-consuming.…”

Detection and Serotyping of Dengue Virus in Serum Samples by Multiplex Reverse Transcriptase PCR-Ligase Detection Reaction Assay

“…More recently this assay has been adapted for the detection of bacterial pathogens in blood cultures (34) and of West Nile virus in serum and mosquito pools (38). Since even a single base mismatch at the ligation junction prevents successful ligation, the technique is highly specific (21). Furthermore, such assays are ideal for multiplexing, since several primer sets can ligate along a DNA template without the interference encountered in purely polymerase-based assays (10,22).…”