Detection of point mutations with a modified ligase chain reaction (Gap-LCR) (original) (raw)

Journal Article

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Abbott Laboratories, Probe Diagnostics Business Unit

Abbott Park, IL 60064, USA

* To whom correspondence should be addressed

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Abbott Laboratories, Probe Diagnostics Business Unit

Abbott Park, IL 60064, USA

Search for other works by this author on:

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Abbott Laboratories, Probe Diagnostics Business Unit

Abbott Park, IL 60064, USA

Search for other works by this author on:

Abbott Laboratories, Probe Diagnostics Business Unit

Abbott Park, IL 60064, USA

Search for other works by this author on:

Received:

04 October 1994

Revision received:

03 January 1995

Accepted:

03 January 1995

Published:

25 February 1995

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Klara Abravaya, John J. Carrino, Sharon Muldoon, Helen H. Lee, Detection of point mutations with a modified ligase chain reaction (Gap-LCR), Nucleic Acids Research, Volume 23, Issue 4, 25 February 1995, Pages 675–682, https://doi.org/10.1093/nar/23.4.675
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Abstract

DNA amplification systems are powerful technologies with the potential to impact a wide range of diagnostic applications. In this study we explored the feasibility and limitations of a modified ligase chain reaction (Gap-LCR) In detection and discrimination of DNAs that differ by a single base. LCR is a DNA amplification technology based on the ligatlon of two pairs of synthetic ollgonucleotldes which hybridize at adjacent positions to complementary strands of a target DNA. Multiple rounds of denaturation, annealing and ligation with a thermostable ligase result in the exponential amplification of the target DNA. A modification of LCR, Gap-LCR was developed to reduce the background generated by target-Independent, blunt-end ligation. In Gap-LCR, DNA polymerase fills in a gap between annealed probes which are subsequently joined by DNA ligase. We have designed synthetic DNA targets with single base pair differences and analyzed them in a system where three common probes plus an allele-speclfic probe were used. A single base mismatch either at the ultimate 3′ end or penultimate 3′ end of the allele specific probe was sufficient for discrimination, though better discrimination was obtained with a mismatch at the penultimate 3′ position. Comparison of Gap-LCR to allele-speclf ic PCR (ASPCR) suggested that Gap-LCR has the advantage of having the additive effect of polymerase and ligase on specificity. As a model system, Gap-LCR was tested on a mutation in the reverse transcriptase gene of HIV, specifically, one of the mutations that confers AZT resistance. Mutant DNA could be detected and discriminated in the presence of up to 10 000-fold excess of wild-type DNA.

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