Automated DNA diagnostics using an ELISA-based oligonucleotide ligation assay.

Abstract

DNA diagnostics, the detection of specific DNA sequences, will play an increasingly important role in medicine as the molecular basis of human disease is defined. Here, we demonstrate an automated, nonisotopic strategy for DNA diagnostics using amplification of target DNA segments by the polymerase chain reaction (PCR) and the discrimination of allelic sequence variants by a colorimetric oligonucleotide ligation assay (OLA). We have applied the automated PCR/OLA procedure to diagnosis ofcommon genetic diseases, such as sickle cell anemia and cystic fibrosis (AF508 mutation), and to genetic linkage mapping of gene segments in the human T-cell receptor f-chain locus. The automated PCR/OLA strategy provides a rapid system for diagnosis of genetic, malignant, and infectious diseases as well as a powerful approach to genetic linkage mapping of chromosomes and forensic DNA typing.

GST, NAT1, CYP1A1 polymorphisms and risk of esophageal and gastric adenocarcinomas

“…The CYP1A1 Ile462Val alleles were identified using an oligonucleotide ligation-based genotyping assay [12]. The PCR primers utilized in this assay were as follows:…”

Parallel molecular genetic analysis

“…Allele-specific mutation/polymorphism detection methods include direct DNA sequence analysis, 98 reverse dot-blot with allele-specific oligonucleotide hybridization, 99 allele-specific PCR amplification, 100 oligonucleotide ligation amplification (OLA), 101 artificial introduction of restriction sites, 102 ligase chain reaction 103 and DNA minisequence analysis. [104][105][106] PCR-OLA has been applied to DNA diagnostics, 107 genetic mapping using biallelic markers 108 and YAC library screening. 109 A variation on PCR-OLA to increase sample throughput makes use of sequencecoded separation.…”

“Spot-On” SNP Genotyping: Figure 1.

“…One approach uses DNA ligase, in which ligation of two allelespecific oligonucleotides occurs in a sequence-dependent manner. This method has been adapted to both solid phase and array based genotyping (Nickerson et al 1990;Gunderson et al 1998;Iannone et al 2000). Allelespecific, enzymatic cleavage of "flap" DNA probes has also been shown to be an effective method (Lyamichev et al 1999).…”