Ligase Chain Reaction (LCR)
Summary
Ligase chain reaction (LCR) is the method which detects a specific nucleotide sequence with some similarities to polymerase chain reaction. It is similar to polymerase chain reaction (PCR) as it uses DNA polymerase and specific primers designed to bind to the target. However, it is unlike to PCR as the primers are not used to amplify the DNA sequence between the primers. In the presence of target sequences, both primers will hybridize in such a way that one primer will lie directly to the second primer of the pair. In the presence of DNA ligase, these two primers are joined to form a single strand and the target sequence will have been effectively duplicated. In the presence of a mutation or if the target sequence is not present, the primers will not bind and ligation will not occur. As the cycle is repeated, only the target sequence (if it is present) is amplified. LCR exploits four primers to obtain only two types of information: (1) presence of adjacent target sequences, and (2) presence of perfect complementarity to the primers at the junction of these sequences. LCR amplification derives the specificity from the initial hybridization of primer to target DNA. This specificity is enhanced by the use of oligonucleotides and temperatures near the oligonucleotide Tm. In LCR there are two important factors which are the design of primers and LCR conditions.
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LCR assays have been developed for the detection of genetic diseases as well as for the detection of bacteria and viruses. In many of these applications, LCR is preceded by an initial PCR step to achieve a greater sensitivity of the respective assays.
Ligase Chain Reaction (LCR)
Introduction
A method of detecting a specific nucleotide sequence with some similarities to polymerase chain reaction. Like PCR, it uses DNA polymerase and specific primers designed to bind to the target. However, unlike PCR, the primers are not used to amplify the DNA sequence between the primers. Instead, if the target sequence is present, both primers will hybridize so that one primer will lie directly adjacent to the second primer of the pair. In the presence of DNA ligase, these two primers are joined to form a single strand and the target sequence will have been effectively duplicated. In the presence of a mutation or if the target sequence is not present, the primers will not bind and ligation will not occur. As the cycle is repeated, only the target sequence (if it is present) is amplified.
Allele-specific LCR employs four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA and a complementary set of adjacent oligonucleotides, which hybridize to the opposite strand. Thermostable DNA ligase will covalently link each set, provided that there is complete complementarity at the junction. Because the oligonucleotide products from one round may serve as substrates during the next round, the signal is amplified exponentially, analogous to PCR amplification. A single-base mismatch at the oligonucleotide junction will not be amplified and is therefore distinguished. A second set of mutant specific oligonucleotides is used in a separate reaction to detect the mutant allele.
History
Since its discovery in 1985, the polymerase chain reaction (PCR) has had a profound impact on detecting genetic and infectious diseases, identifying new genes, and unraveling the mysteries of protein-ligand recognition. Its universal utility is due to the specificity of amplification and ease of cycling made possible by the cloning and careful characterization of a thermostable polymerase from Thermus aquaticus. Likewise, cloning of a thermostable ligase enabled a new amplification method, termed ligase chain reaction (LCR), to both amplify DNA and discriminate a single base mutation. Although these DNA amplification techniques are new, they bring to fruition the “enzymes as reagents” philosophy expounded by A. Kornberg and I.R. Lehman a quarter of a century ago.
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Principle of Ligase Chain Reaction
The principle of LCR is based in part on the ligation of two adjacent synthetic oligonucleotide primers, which uniquely hybridize to one strand of the target DNA. The junction of the two primers is usually positioned so that the nucleotide at the 3′ end of the upstream primer coincides with a potential single base-pair difference in the targeted sequence. This single base-pair difference may define two different alleles, species, or other polymorphisms correlated to a given phenotype. If the target nucleotide at that site complements the nucleotide at the 3′ end of the upstream primer, the two adjoining primers can be covalently joined by the ligase. The unique feature of LCR is a second pair of primers, almost entirely complementary to the first pair, that are designed with the nucleotide at the 3′ end of the upstream primer denoting the sequence difference. In a cycling reaction, using a thermostable DNA ligase, both ligated products can then serve as templates for the next reaction cycle, leading to an exponential amplification process analogous to PCR amplification. If there is a mismatch at the primer junction, it will be discriminated against by thermostable ligase and the primers will not be ligated. The absence of the ligated product therefore indicates at least a single base-pair change in the target sequence.
Ligase Chain Reaction (LCR)
Probes hybridise specifically to the target. The probes are ligated to form a copy of the target DNA. Reaction is heated to separate DNA strands. ss DNA forms new templates for further probe binding.
Specificity, Thermostability, and Thermophilic Organisms
LCR exploits four primers to obtain only two types of information: (1) presence of adjacent target sequences, and (2) presence of perfect complementarity to the primers at the junction of these sequences.
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LCR amplification derives the specificity from the initial hybridization of primer to target DNA. This specificity is enhanced by:
• Use of oligonucleotides of sufficient length to uniquely identify individual humans or the target genome, and
• Use of temperatures near the oligonucleotide Tm.
With PCR, background target-independent amplification results in primer dimers, which are of lower molecular weight and thus easily distinguished. However, with LCR, background target-independent amplification yields the same size product. Hence, to reduce LCR to practice, it was necessary to eliminate target independent ligations completely. This was accomplished with use of a thermostable ligase.
For an effective amplification reaction to take place, a thermostable enzyme must not become denatured irreversibly when subjected to the elevated temperatures (about 90-100ºC) for the amount of time necessary to effect complete denaturation of double stranded DNA (about 30-60 sec).
Both Taq polymerase and Taq ligase retain activity after 20 or 30, or more, repeated 1-min exposures to 94ºC and hence are termed thermostable. The TaqI restriction endonuclease, isolated from the same thermophilic T. aquaticus species, does not survive such treatment (being completely inactivated after 20 min at 85ºC and hence is termed a thermophilic enzyme.
T. aquaticus YT1 and T. thermophilus HB8 were isolated originally on two different continents. Currently, they are classifed by the American Type Culture Collection (ATCC) as a single species. The amino acid sequences of T. aquaticus YT1 and T. thermophilus HB8 restriction endonucleases, methylases, and DNA polymerases show 77, 79, and 88% identity, respectively, and DNA homology studies of numerous thermophilic isolates suggest that these organisms may indeed be separate species. Until the taxonomy is resolved, both designations are correct and will be used interchangeably.
Important Factors for LCR Reactions
Accurate results from LCR assays depend on a variety of factors, including primer design and reaction conditions. Based on our experience and those of others over the past 3 years, a few of the most important factors that need to be considered in the development of LCR assays follow.
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Design of LCR Primers
To minimize target-independent ligation, LCR primers with a single base-pair overhang, rather than blunt ends, should be used. The importance of single base-pair overhangs is shown by Kalin who reported a relatively high amount of target-independent ligation using primers with blunt ends. The Tm of all four primers of one set of LCR primers should be within a narrow temperature range, ideally with an absolute Tm of 70ºC ± 2ºC Furthermore, the primers should be designed so that one primer cannot serve as a bridging template for other primers and therefore lead to target-independent ligation.
Adding noncomplementary tails of two nucleotides or longer to the nonadjacent 5′ ends of the primers should prevent ligation of the 3′ ends. Depending on the discriminated nucleotides, different amounts of ligation product are observed with a mismatched target. Expected amounts of false ligation for specific mismatches are shown in Table. These data can be used for designing primers with the lowest possible rate of false ligation when some choice between different target sequences exists.
Table: Noise-to-signal ratio for certain mismatches in the LCR
Oligonucleotide base-target base Noise-to-signal ratio a (%)
A-A, T-T 1.1
T-T, A-A
