High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation

Abstract

We describe a non-isotopic, semi-automated method for large-scale multiplex analysis of nucleic acid sequences, using the cystic fibrosis transmembrane regulator (CFTR) gene as an example. Products of a multiplex oligonucleotide ligation assay (OLA) are resolved electrophoretically from one another and from unligated probes under denaturing conditions with fluorescence detection. One ligation probe for each OLA target carries a fluorescent tag, while the other probe carries an oligomeric non-nucleotide mobility modifier. Each OLA product has a unique electrophoretic mobility determined by the ligated oligonucleotides and the mobility-modifier oligomer arbitrarily assigned (coded) to its target. The mobility range for practical mobility modifiers is much wider than the accessible range from unmodified ligated oligonucleotides of practical length. Each mobility modifier is built from phosphoramidite monomers in a stepwise manner on its associated oligonucleotide using an automated synthesizer. The resulting mobility modifiers lower the probe-target duplex Tm by less than 3 degrees C and retard probe-target annealing by less than 50%, with negligible effect on OLA yield and specificity. This method is especially useful for allelic discrimination in highly polymorphic genes such as CFTR.

A review on SNP and other types of molecular markers and their use in animal genetics

“…Detection of the alleles can be performed directly in the microplate wells by colorimetric approaches [76]. Multiplexing and the use of gel separation has also been described [28].…”

Improving the Fidelity of Thermus Thermophilus DNA Ligase

“…Our laboratory has cloned the thermostable Thermus thermophilus DNA ligase (Tth DNA ligase), which we and others have used in the ligase chain reaction (LCR) and ligase detection reaction (LDR) for detecting infectious agents and genetic diseases (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). The success of these and future disease detection assays, such as identifying tumor-associated mutations in an excess of normal DNA, depend on the exquisite fidelity of Tth DNA ligase.…”

Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors

“…The optimal multiplex detection scheme involves a primary PCR ampli®cation, followed by either LDR (two primers, same strand) or LCR (four primers, both strands) detection. This approach has been successfully used to detect multiple mutations in 21-hydroxylase de®ciency (Day et al, 1995(Day et al, , 1996, hyperkalemic periodic paralysis (Feero et al, 1993), human allele polymorphisms (Belgrader et al, 1996) and cystic ®brosis (Eggerding et al, 1995;Grossman et al, 1994;Winn-Deen et al, 1993). The thermostable ligase and engineered mutants exhibit high ®delity in discriminating all possible matched and mismatched base pairs .…”