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Ligase Chain Reaction 135
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Ligase Chain Reaction
William H. Benjamin, Jr., Kim R. Smith, and Ken B. Waites
1. Introduction
The ligase chain reaction (LCR) is one of many techniques developed in recent years
to detect specific nucleic acid sequences by amplification of nucleic acid targets. The
LCR has been used for genotyping studies to detect tumors and identify the presence
of specific genetic disorders such as sickle cell disease caused by known nucleotide
changes that occur as a result of point mutations and has now become widely used
in infectious disease detection, both in the diagnostic and research settings, primarily
focusing on infections caused by microbes that have proven difficult to detect by
traditional culture techniques. The LCR is now recognized as the method of choice
for detection of urogenital infections due to Chlamydia trachomatis because of its
greater sensitivity as compared to traditional cell culture or nonamplified DNA probes
or antigen-detection assays. Other uses of the LCR have also been reported (1–8). When
used for detection of infectious diseases, amplification tests such as the LCR have the
additional advantages in that they do not require viable organisms in a specimen, a
single specimen can be used to detect multiple different pathogens, provided suitable
primers are available, and easily obtained specimens such as urine can be used for
diagnostic purposes, making screening of large numbers of persons practical, as well as
facilitating research to better understand the epidemiology of specific diseases.
Ligation of adjacent oligonucleotides while hybridized to a template was first
investigated by Besmer et al. in 1972 (9). The first use of ligases to join oligonucleotides
as a means to differentiate sequence variants was reported in 1988 (10,11) and the first
“real” LCR utilizing cyclical denaturation hybridization and ligation of two pairs of
oligomers was described in 1989 (12). Thermostable ligases that eliminate the necessity
of adding ligase after each denaturation step were introduced by Barany in 1991 (1).
Simple LCR consists of two complementary oligonucleotide pairs (four oligonucleotides
20–35 nucleotides each) that are homologous to adjacent sequences on the
target DNA, as opposed to two used in the polymerase chain reaction (PCR) assay.
The adjacent pairs are ligated when they hybridize to the complementary sequence
next to each other in a 3′ to 5′ orientation on the same strand of the target DNA.
The 5′ nucleotide of the ends of the primers to be ligated must be phosphorylated.
Newly ligated oligonucleotides become targets in subsequent cycles so logarithmic
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
135