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The Ligase Chain Reaction in a PCR World

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The Ligase Chain Reaction in a PCR World Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press The Ligase Chain Reaction in a PCR World Francis Barany Department of Microbiology, Hearst Microbiology Research Center, Cornell University Medical College, New York, NY 10021 Since its discovery in 1985, the tion of single-base substitutions in amount of time necessary to effect polymerase chain reaction (PCR) has DNA diagnostics; (4) characterization complete denaturation of double- had a profound impact on detecting of DNA ligases; (5) cloning of DNA stranded DNA (about 30-60 sec). Both genetic and infectious diseases, identi- ligases; (6) use of ligases in DNA detec- Taq polymerase and Taq ligase retain fying new genes, and unraveling the tion; (7) other methods that use ligase, activity after 20 or 30, or more, mysteries of protein-ligand recogni- such as ligase-mediated PCR and the repeated 1-min exposures to 94~ (6,8) tion.(1-s) Its universal utility is due to branch capture reaction; and (8) poten- and hence are termed thermostable. The the exquisite specificity of amplifica- tial new uses of ligase in PLCR and TaqI restriction endonuclease, iso- tion and ease of cycling made possible nested LCR amplification reactions. It lated from the same thermophilic T. by the cloning and careful character- will close with speculations on future aquaticus species, does not survive such ization of a thermostable polymerase prospects. treatment (being completely in- from Thermus aquaticus. (6,7) Likewise, activated after 20 min at 85~ cloning of a thermostable ligase SPECIFICITY, THERMOSTABILITY, and hence is termed a thermophilic en- enabled a new amplification method, AND THERMOPHILIC ORGANISMS zyme. termed ligase chain reaction (LCR), to PCR amplification exploits two primers T. aquaticus YT102) and T. both amplify DNA and discriminate a to obtain three types of information: thermophilus HB8 (13) were isolated single base mutation. (8-1~ Although (1) presence of target sequence, (2) dis- originally on two different continents. these DNA amplification techniques tance between primers, and (3) se- Currently, they are classifed by the are new, they bring to fruition the "en- quence in between the primers. LCR American Type Culture Collection zymes as reagents" philosophy ex- exploits four primers to obtain only (ATCC) as a single species. (14) The pounded by A. Kornberg and I.R. Leh- two types of information: (1) presence amino acid sequences of T. aquaticus man a quarter of a century ago. of adjacent target sequences, and (2) YT1 and T. thermophilus HB8 restric- Allele-specific LCR employs four presence of perfect complementarity to tion endonucleases,O5-17) methyl- oligonucleotides, two adjacent oligo- the primers at the junction of these se- ases, (ls-17) and DNA polymerases (18) nucleotides which uniquely hybridize quences. Both LCR and PCR amplifica- show 77, 79, and 88% identity, respec- to one strand of target DNA and a tion derive their specificity from the tively, and DNA homology studies of complementary set of adjacent oligo- initial hybridization of primer to target numerous thermophilic isolates sug- nucleotides, which hybridize to the op- DNA. This specificity is enhanced by: gest that these organisms may indeed posite strand (see Fig. 1, top left). (1) use of oligonucleotides of sufficient be separate species.Og) Until the Thermostable DNA ligase will covalent- length to uniquely identify individual taxonomy is resolved, both designa- ly link each set, provided that there is humans or the target genome, and (2) tions are correct and will be used inter- complete complementarity at the junc~ use of temperatures near the oligo- changeably. tion. (8) Because the oligonucleotide nucleotide T m. With PCR, background products from one round may serve as target-independent amplification re- DNA DIAGNOSTICS substrates during the next round, the sults in primer dimers, which are of DNA diagnostics employs the tools of signal is amplified exponentially, lower molecular weight and thus easily molecular biology to detect the analogous to PCR amplification. A distinguished. However, with LCR, presence of bacterial and viral in- single-base mismatch at the oligo- background target-independent ampli- fectious agents, genetic traits, and dis- nucleotide junction will not be ampli- fication yields the same size product. eases. (2~ Furthermore, DNA diag- fied and is therefore distinguished (Fig. Hence, to reduce LCR to practice, it nostics can uniquely identify humans, 1, top right). A second set of mutant- was necessary to eliminate target inde- animals, and plants. (2~ This often specific oligonucleotides is used in a pendent ligations completely. This was ac- demands exquisite specificity to dis- separate reaction to detect the mutant complished with use of a thermostable tinguish closely related (drug-resistant allele. ligase.(8-10) vs. -sensitive) pathogens or single-allele This review will give a brief intro- For an effective amplification reac- diseases, which may consist of subtle duction to (1) determinants of tion to take place, a thermostable en- deletions, insertions, or single- specificity in amplification reactions; zyme must not become denatured irre- nucleotide substitutions in over 3 bil- (2) differences between thermostable versibly when subjected to the elevated lion base pairs of target DNA. A reliable and thermophilic enzymes; (3) detec- temperatures (about 90-100~ for the DNA diagnostics method requires 1:5-169 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/91 $3.00 PCR Methods and Applications 5 Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press trophoresis, (26) RNase or chemical cleavage of mismatched heterodup- lexes, (27-29) incorporation of biotiny- lated mononucleotides into single- stranded DNA, (3~ fluorescence PCR amplification/detection, (31) allelic- specific PCR using nested PCR, (32,33) or polymerase amplification of specific al- leles (PASA). (34-s6) Ligase-based assays, including LCR (which accomplishes both amplification and single- nucleotide discrimination in the same step) will be discussed below. CHARACTERIZATION OF DNA LIGASES In the late 1960s, models of DNA recombination, replication, and repair proposed the existence of an enzyme that could form a covalent phosphate link between two strands of DNA. No less than five separate groups, using five separate assays, independently and virtually simultaneously, announced the discovery of such an enzyme, termed DNA ligase.(s7-44) DNA ligase uses either an ATP (T4 enzyme) or NAD (Escherichia coli enzyme) cofactor to join covalently the adjacent 3' hydroxyl and 5' phosphoryl termini of nucleotides that are perfectly hydrogen bonded to a complementary strand. Substrate assays for this reaction in- FIGURE 1 (Upper) Allele-specific DNA amplification and detection using the ligase chain clude: (1) circularization of linear reaction (LCR). DNA is heat-denatured (94~ and four complementary oligonucleotides an- "sticky end" phage X DNA to a form neal to the target at a temperature near their melting temperature (65~ Tin). Thermostable that does not denature at alkaline ligase will covalently attach only adjacent oligonucleotides that are perfectly complementary pH, (37,38) (2) resistance of 5'32p- to the target (left). The products from one round of ligations become the targets for the next labeled oligo(dTlso) to alkaline phos- round, and thus the products increase exponentially. Oligonucleotides containing a single- phatase treatment after ligation in the base mismatch at the junction do not ligate efficiently, and therefore do not amplify product presence of complementary strand (right). The diagnostic oligonucleotides (striped strands) have the discriminating nucleotide on poly(dA4000), (39,4~ (3) covalent capture the 3 ' end for both the top and bottom strands. Thus, target-independent, four-way ligation and alkaline-resistant precipitation of would require sealing an (unfavorable) single-base 3' overhang. (Lower) Nucleotide sequence and corresponding translated sequence of the oligonucleotides used in detecting ~A and ~S labeled oligo(dC) after ligation to globin genes. Oligonucleotides 101 and 104 detect the ~A target, while oligonucleotides 102 cellulose-oligo(dC) in the presence of and 105 detect the BS target when ligated to labeled oligonucleotides 107 and 109, respec- complementary poly(dI), (41) and (4) tively. The diagnostic oligonucleotides (101, 102, 104, and 105) contained slightly different resistance of labeled poly(dAT) to ex- length tails to minimize aberrant ligation products and to facilitate discrimination of various onuclease III degradation after forming products when separated on a polyacrylamide denaturing gel. Oligonucleotides have calcu- a self-complementary closed circular lated Tm values of 66~ to 70~ (calculated as described in ref. 107, or for a more precise loop. (4s) The ligation reaction occurs in determination see ref. 105), just at or slightly above the ligation temperature. (Adapted from three discrete and reversible steps: (1) ref. 8.) formation of a high-energy enzyme in- termediate by transfer of the adenosyl faithful amplification of target se- (3SR), (21) or Q-~ replicase-mediated group from NAD (or ATP) to the E- quences with no false positives, ac- RNA amplification.(22) Subsequently, amino group of a lysine residue; (2) curate single-base discrimination, low small deletions, insertions, or single- transfer of the adenosyl group to the background,
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