The Ligase Chain Reaction in a PCR World

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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 amplification 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

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trophoresis, (26) RNase or chemical cleavage of mismatched heterodup- lexes, (27-29) incorporation of biotinylated mononucleotides into single- stranded DNA, (3~ fluorescence PCR amplification/detection, (31) allelic- specific PCR using nested PCR, (32,33) or polymerase amplification of specific alleles (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 intermediate 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, and either complete auto- base mismatches may be detected 5'phosphate of one DNA strand thus mation or simple inexpensive kits. The through use of allele-specific oligo- forming an activated pyrophosphate initial target nucleic acid amplification nucleotide hybridization,(2a,24) reverse- linkage; and finally (3) attack of this may be accomplished using the PCR, (6) oligonucleotide blot-dot hybridiza- activated 5'end by a 3'-hydroxyl self-sustained sequence replication tion, (2s) denaturing gradient gel elec- group on the adjacent DNA strand,

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thus forming a phosphodiester link be- pact that cloning the actual T4 and E. units/rag. (62) DNA sequence analysis of tween the two DNA strands, and coli ligase genes themselves had on the the T. thermophilus ligase gene revealed eliminating AMP. (46-s4) These steps are early days of biotechnology. This clon- a single chain of 676 amino acid shown below for the E. coli DNA ligase. ing has been greatly facilitated by the residues with 47% identity and 66% characterization of two temperature- similarity to the E. coli ligase gene. (62) E-(lys)-NH 2 + AMP-P-N + sensitive mutants of the E. coli A similar thermophilic gene also has E-(lys)-NH2+ -AMP + NMN (1) gene. ~63-6s) The ligts4 strain contained been cloned by screening for a heat- normal levels of enzyme at 25~ and stable, ligase-adenylate inter- E-(lys)-NH2+ -AMP + 5' P-Oligo#2 only 1% at 42~ whereas the ligts7 mediate, (77) essentially as described AMP-P-Oligo#2 + E-(Iys)-NH 2 (2) strain had only 1% enzyme at 25oc, previously for the yeast ligase and lost viability at 42~ Neither genes.(S6, s7) Oligo#1-3 ' OH + AMP-P-Oligo#2 strain supported growth of an Oligo#1-P-Oligo#2 + AMP (3) integration-deficient (Red-) ~. phage, LIGASE-MEDIATED DNA DETECTION and the combination of this From the variety of exceedingly sensi- The ligase-adenylate intermediate host-vector system provided an tive assays first described for ligase, it was so stable that it could be readily elegant positive selection of recom- was readily apparent that this enzyme identified and separated by acid precip- binant phages containing the T4 or E. could serve as a reporter for the itation of radioactively labeled en- coli ligase gene. (60-68) Overproduction presence of two adjacent strands of zyme, gel filtration, or its slower of the enzyme reduced purification to DNA hybridized to a complementary mobility after electrophoresis through a simple three-step procedure. (69,7~ target DNA strand.(37-41,4s) As part of a an SDS-polyacrylamide gel. (47,49,s~ DNA sequence analysis of the E. coli herculean effort to achieve total Proteolytic degradation of the adenyl- gene revealed a single chain of 671 synthesis of tRNA genes, the adjacent ated adduct revealed adenosine 5' amino acid residues, (71) with no sig- position of two oligonucleotides could monophosphate linked to the E-amino nificant identities to the T7, T4, or be proved by the ability of T4 group of lysine. (46) This phosphamide yeast ligases. (so,s 7, 72,73) ligase to link these oligonucleotides linkage could be reversed by exchang- Thermostable ligase was cloned by only when hybridized to the r strand ing with NMN or PPi in the absence of screening for growth of a ligts7 deriva- of ~b8Opsu+mDNA.(78) A dozen years DNA substrate, or alternatively trans- tive (AK76) at 42~ when comple- passed before synthetic oligonucleo- ferred to the 5'phosphate of a DNA mented with plasmid libraries of T. tides were readily available, allowing substrate. Formation of a ligase-aden- aquaticus HB8 DNA. (SA~ To assure that for practical use of this concept. This ylate intermediate has been used to the highly methylated T. aquaticus HB8 assay has been used to detect the identify and clone two yeast ligase DNA (including TCGA and AATI" presence of minute quantities of ligase genes.(S s-sT) sitesOS)) did not undergo mrr-assoc- in crude preparations,(79) or to detect The T4 and E. coli enzymes exhibit iated restriction or mutation, (74) it was the presence of sub-picomole quant- slightly different activities; only the E. necessary to prepare libraries in an E. ities of ~. phage DNA, and has clear coli enzyme is activated by ammonium coli host strain (such as AK76) lacking diagnostic implications.(8~ Land- ions (about 20-fold), whereas the T4 not only mrrA, but, more importantly, egren et al. pioneered the use of such enzyme has higher activity for blunt a newly discovered methyl-dependent an oligonucleotide ligation assay (OLA) ends or RNA-DNA hybrids.(42) Blunt- endonuclease termed mrrB. (8,15,62) True to detect single nucleotide substitu- end activity may be readily detected complementation by a plasmid con- tions in both cloned and clinical for the E. coli enzyme when using taining the thermostable ligase gene samples. (59,82-89) Use of a biotin "hook" molecular-crowding agents such as (pDZ1) could be distinguished from on the first probe and a suitable non- PEG, (sS) or appropriately sensitive as- revertants by pinpoint colony size, as isotopic reporter group on the second says. (sg) T. thermophilus HB8 ligase well as presence of a thermostable probe allowed for product capture shows only a very slight stimulation by NAD-dependent nick-closing (DNA (with streptavidin) and detection, thus ammonium ions, (6~ and like its E. coli ligase) activity in crude extracts when circumventing the need for elec- homolog, displays blunt-end activity assayed at 65~ Furthermore, DNA se- trophoresis or precise hybridization in the presence of PEG, even at quence analysis of the first 60 codons conditions. (82) The method has been 65~ (61) As observed with the E. coli of the putative gene revealed >50% combined with a primary PCR amplifi- enzyme, the thermostable ligase-aden- amino acid identity to E. coli cation to screen for sickle cell anemia, ylate intermediate migrates with an ap- ligase. (8,62) Enzyme overproduction cystic fibrosis, ctl-antitrypsin defi- parent molecular weight of about was achieved by replacing the ciency, and T-cell receptor polymorph- 81,000, as compared with about 78,000 endogenous transcription and trans- isms in an automated ELISA-based for the nonadenylated form on an SDS- lation signals with a phoA pro- format.(83, 8s) polyacrylamide gel.(62) moter-ribosome binding site. (62,7s) A Thermostable ligase discriminated heat treatment step during purification single-base mismatches under both CLONING OF DNA LIGASES (65~ for 20 min (70)) allowed for rapid LDR (_ligase detection reaction; using Most molecular biologists use huge ex- production of exo- and endonuclease- two adjacent probes) and LCR (_ligase cesses of DNA ligase for cloning count- free thermostable ligase with a specific chain reaction; using two pairs of ad- less exotic genes, unaware of the im- activity of 1.6 million (nick-closing) jacent probes) conditions, with a

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signal-to-noise ratio ranging from 75 than for ~A-specific oligonucleotides, tached only to these newly synthesized to greater than 500. (8) With no back- when either plasmid or human chrom- blunt ends with T4 ligase. Both a sec- ground over a range of salt conditions, osomal target DNA was used, with ei- ond primer, longer and downstream this represented a substantial improve- ther radioactive (a) or fluorescent detec- from the first, and the unidirectional ment in specificity, sensitivity, and tion. (92) This difference may be a func- linker primer are then used to amplify flexibility when compared with use of tion of the exact nucleotide sequence the appropriate fragments specifically. mesophilic enzymes. (s9,87,88,90) Fur- at the ligation junction, and, although Products are identified by either thermore, for amplification reactions, subtle, is amplified multiple times. Southern blotting or by extending mesophilic ligases were unable to Thus, about two- to threefold more with a third end-labeled primer, and eliminate target-independent (blunt product was detected for ~A than for ~s fragments are analyzed on a denatur- end) background ligation, thus limit- target DNA. Such differences should be ing poyacrylamide sequencing gel. This ing practical use. (s9,88,90) Four-way considered when designing assays method has been successfully applied (target-independent) ligation was mini- wherein two allele-specific oligo- to genomic sequencing, (97) genomic mized in LCR with thermostable ligase nucleotides compete for ligation to an DMS footprinting, (98) genomic by the following procedures: (1) addi- adjacent invariant oligonucleotide. Op- methylation studies, (97,99,1~176and clon- tion of 4 ,g of carrier salmon sperm timized LCR conditions for detecting ing of promoter elements.O01) For DNA, (2) use of single-base 3' over- sickle cell anemia, Mycobacterium cloning purposes, use of a non- hangs on discriminating oligonucleo- tuberculosis, and human papil- complementary unidirectional linker tides, (3) 5' phosphorylation of the ad- lomaviruses are presented in Table (i.e., lacking a palindromic restriction jacent (ligating) oligonucleotides only, 1 .(8,92-94) site on the 3'end) or a vectorette- (4) presence of noncomplementary linker with an internal "bubble", (1~ tails on the outside of olignucleotides, LIGASE-MEDIATED PCR AND THE would probably reduce formation of and (5) use of cycling conditions at or BRANCH-CAPTURE REACTION aberrant PCR products. near the oligonucleotide Tm.(a) Omis- Ligase-mediated PCR combines the In a branch-capture reaction (BCR), sion of either carrier salmon sperm specificity of hybridizing to a single a displacer strand is used to bring a DNA or use of "blunt-end" oligonucleo- primer site with (nonspecific) blunt- complementary recipient duplex tides gave detectable background sig- end linker ligation to amplify and strand 3' end adjacent to the 5' end of nal. (8,91) The method could detect 200 identify sequences where only one side a complementary linker strand, creat- target molecules, and could dis- is known. (9s,96) Chromosomal DNA is ing a substrate for covalent linkage by criminate between normal 13A- and nicked or cleaved in a defined way, the ligase. O~176 Use of 5Met-dCTP in an sickle ~S-globin genotypes from l O-~tl DNA denatured, and a specific primer asymmetrically synthesized displacer blood samples, as shown in Figure 2.(8) extended with Sequenase to generate a and thermostable ligase greatly en- The efficiency of ligation (and hence new blunt end. A unidirectional (stag- hanced the specificity of DNA cap- detection) was somewhat less for ~s. gered) unphosphorylated linker is at- ture.O04) The branch-capture reaction may also be used as an alternative to biotin/streptavidin as a means of capturing specific LCR-, OLA-, or PCR- generated product for detection.(8,2~176 Combining the branch-capture reaction (steps 1-3, Fig. 3) with ligation-mediated PCR (steps 4-8, Fig. 3) provides the potential for specifically capturing and amplifying any chromosomal DNA fragment.

PLCR, NESTED LCR, AND OTHER VARIATIONS LCR amplification aims to discriminate accurately between different alleles while achieving the highest signal-to- FIGURE 2 Autoradiogram showing detection of ~-globin alleles in human genomic DNA. noise ratio. For example, LCR did not DNA was isolated from blood samples from normal (~A~A), carrier ([3A[3S), and sickle cell amplify a T-T, G-T, C-T, or C-A 3' ter- ([3S~s) individuals as described in ref. 8. Genomic DNA (corresponding to 10 ~tl of blood, or minal mismatch, (8) as has been about 6 • 104 nucleated cells) was tested in two separate tubes containing labeled reported for some allele-specific PCR oligonucleotides (107 and 109; 200,000 cpm -- 40 fmoles each), and either unlabeled 13A test amplifications. (32) One approach to oligonucleotides (101 and 104) or unlabeled 13s test oligonucleotides (102 and 105; 40 fmoles each). Both reaction mixtures were incubated under the same buffer (without salmon sperm increase the specificity of allele-specific DNA), enzyme, and cycle conditions described in method 1 (Table 1). Samples were elec- PCR is to use low (1 ~tM) concentra- trophoresed in a 10% polyacrylamide denaturing gel, and subjected to gel autoradiography tions of dNTPs, although this would overnight, as described in ref. 8. Ligation products of 45 and 46 or 47 and 48 nucleotides in- limit the extent of amplification.OS) dicate the presence of the 13A or 13s globin gene, respectively. (From ref. 8, with permission.) Allele-specific addition of one or two

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TABLE 1 Ligase Chain Reaction Methods

Method I a Method 2 a Method 3 a

Target DNA ~A,~S ~A,~S human papillomavirus Standard detection 1-10 attomoles (6 x 105 105-106 molecules 106 molecules to 6 • 106 molecules) Signal-to-noise 1700 to >2000b 20-30 26 no target under standard conditions single-base mismatch 75 to >500 b 18-30 not done under standard conditions Lowest detection 200 molecules 1000 molecules 500 molecules Position of 3 ' base of both 3' base of both not done discriminating strands (single- strands (single- nucleotide base 3' overhang) base 3' overhang) T m discrimination 64oc-68oc 62oc-64~ not reported oligonucleotides (23- to 28-mers) (20- and 21-mers) T m adjacent 70~ 60~176 not reported oligonucleotides (22-mers) (19- and 20-mers) Amount of each 40 femtomoles 0.5-1 picomole 1-1.5 picomole oligonucleotide (0.28 ng) (3.5-7 ng) (7-10 ng) Volume 10 ~tl 100 B1 50 gl Buffer conditions 20 mM Tris-HC1, pH 7.6 c 20 mM Tris-HCl, pH 7.6 50 mM EPPS, pH 7.8 100 mM or 150 mM KC1 100 mM KCI 100 mM K (as OH and CI-) 10 mM MgC12 10 mM MgCl 2 10 mM MgC12 10 mM DTT 10 mM DTF 1 mM DTF 10 mM NAD § 1 mM NAD + 0.1 mM NAD+ 1 mM EDTA 0.1% Triton X-100 10 mM NH4CI 10 gg/ml BSA Carrier DNA to 4 I~g of salmon sperm DNA 4 ~tg of herring sperm DNA not reported suppress background Additional features 5' phosphate on adjacent fluorescent dye label on fluorescent and for suppression of oligonucleotides only; nonligating ends of all biotin moieties on target independent noncomplementary tails on four oligonucleotides; nonligating ends background outside of oligonucleotides; single-base 3' overhang single-base 3' overhang on on discriminating discriminating oligonucleotides oligonucleotides Thermostable enzyme 15 nick-closing units d 15 nick-closing units not reported Cycle conditions 94~ 1 min 94~ 1 min 85~ 0.5 rain 65~ 4 min 62~ 2 rain 50~ 0.35 min 20 or 30 cycles (5-sec autoextend/cycle) 30 or 35 cycles 30 or 40 cycles or 94~ 0.5 min 65~ 2 rain 30 or 40 cycles

a Method 1 (8) used radioactive detection with direct quantitation of products. Method 2 (92) and method 3 (94) used fluorescent detection essentially as described by Landegren et al. (82) Method 2 has also been applied to detection of Mycobacterium tuberculosis.(93) b Signal-to-noise ratios may be better than indicated for method 1, since no incorrect product was observed at the limit of detection.(8) c pH value determined at 25~ d One nick-closing unit of ligase is defined as the amount of ligase that circularizes 0.5 ~tg of DNase I-nicked pUC4KIXX DNA (about 10-20 nicks per plasmid) in 20 ~1 of 20 mM Tris-HC1, pH 7.6 (at 25~ containing SO mM KC1, 10 mM MgC12, 1 mM EDTA, 10 mM NAD§ 10 mM dithiothreitol, overlaying with a drop of mineral oil, after a 15-min incubation at 65~ (8)

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independent ligation because the oligonucleotide pairs are no longer adjacent to each other. An alternative approach for further improving signal-to-noise is use of a primary LCR amplification to generate the primers required for a secondary allele-specific LCR amplification (nested LCR, see Fig. 5). Two separate tubes are used in the primary amplification, each containing shorter LCR primers (about 15 nucleotides in length, with T m values around 55~ calculated as described in ref. 107, or for a more precise determination see ref. 105) corresponding to about 30 nucleotides flanking the left and about 30 nucleotides flanking the right of the allele-specific base pair, respectively. LCR amplification (cycling between 55~ and 94~ will form significant full-length primer products (30 nucleotides) only in the presence of target DNA. The resultant primers are combined, and fresh ligase and an organic solvent such as ethylene glycol, glycerol, formamide, or DMSO is added (depressing oligonucleotide primer Tm values to about 85~ A second LCR amplification (cycling between 85~ and 94~ will specifically form a full- length product (60-mer) only if the correct allele target DNA is present. Nested LCR should further minimize FIGURE 3 A diagram depicting use of a combination of the branch-capture reaction and target-independent product formation. ligase-mediated PCR to clone any chromosomal DNA fragment (BCLPCR). After cleaving Thermostable ligase may also be chromosomal DNA (lightly shaded strands) with one or more restriction endonucleases used to covalently link two members (preferably generating a 3' overhang), a displacer strand (white strand), partially com- of a hexamer oligonucleotide library to plementary to the end of the recipient strand, and partially complementary to a linker form specific dodecamers for directed strand, and a biotinylated linker (black strand) complementary to the displacer is added. The sequencing of cosmids and other large displacer strand may be either chemically synthesized, or generated via asymmetric PCR, and DNAs.(110-113) Initially, randomly contains modified cytosines (5MedC or 5BrdC) to increase the oligonucleotide Tm above that of the recipient displaced strand. By incubating the reaction at 65~ in the presence of selected nonamers would be used to thermostable ligase, the displacer strand should specifically direct capture of only com- start sequencing randomly throughout plementary recipient strands by covalent linkage to the linker. Upon extending the displacer a cosmid clone.(11~ By judicious strand with Sequenase (darkly shaded strand), the newly generated blunt end provides a choice of two or four hexamers from a suitable substrate for attachment of an unphosphorylated unidirectional linker (striped 2994-member library, one could gener- strands). Excess linker, chromosomal DNA, and enzymes are conveniently removed by cap- ate a dodecamer primer through either turing the biotinylated DNA fragment with streptavidin-coated magnetic particles (Dynal), target-dependent LDR amplification, or and washing in O. 1 N NaOH. After neutralization, a second specific primer (horizontal striped target-dependent or -independent LCR strand) slightly 3' to the displacer oligonucleotide is annealed and extended with Taq amplification using 2-base 3' over- polymerase. Addition of the longer unidirectional linker oligonucleotide followed by PCR hangs among the four oligonucleo- will specifically amplify the desired fragment. (Adapted from refs. 95, 96, and 103-105.) tides.Oll,l14,11s) Improper priming may be minimized by: (1) using Taq polymerase to extend dodecamer nucleotides using the Taq polymerase two oligonucleotides, which would be primers at 37 or 45~ temperatures Stoffel fragment (lacking not only the sealed with Taq ligase, in a process well above the hexamer Tm; and (2) 3'-5'proofreading exonuclease, but termed PLCR (see Fig. 4). This would using one hexamer primer with a also the S'-3'nick-translation ex- simultaneously increase allele-specific 2',3'dideoxynucleotide to block exonuclease (1~ could fill a gap between discrimination while avoiding target- tension off the incorrect LCR dodeca- r O PCR Methods and Applications Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press

single base pair. For example, among the infectious diseases, it is important to differentiate pathogenic agents in the presence of nonpathogenic normal flora, or to detect emerging viral sub- populations where the mutations are known, such as the multiple mutations in HIV conferring resistance to AZT. (124) LCR amplification currently has a signal-to-noise ratio of from 75 to greater than 500, (8) and can thus theoretically detect a pathogenic or resistant subpopulation of from 7% to 1% with a fivefold higher signal than for the nonpathogenic or sensitive population signal. Whether LCR will tolerate internal mismatches between the primers and (HIV) viral variants, (32A2s) while still maintaining high signal-to-noise ratios, remains to be determined. One potential roadblock in devel- FIGURE 4 Allele-specificDNA amplification and detection using the Taq polymerase Stoffel oping prenatal diagnosis for fragment and Taq ligase (PLCR). DNA is heat-denatured (94~ and four complementary (but monogenic diseases is the large variety not adjacent) oligonucleotides anneal to the target at a temperature near their melting of mutations present in the human temperature (65~ The diagnostic oligonucleotides (striped strands) contain the discriminat- population. As of 1990, over 90 point ing nucleotide on the 3' end. The Taq polymerase Stoffel fragment, which lacks the 5 ' -3 ' mutations producing B-thalassemia exonuclease, is added in the presence of 1 ~M dGTP and dCTP to fill the gap between have been catalogued, and these in- oligonucleotides (black), provided the last nucleotide is complementary to the target DNA. clude frameshift mutations, nonsense Thermostable ligase would subsequently covalently link only the extended diagnostic codons, small deletions, transcription oligonucleotide to the newly adjacent (white strand) oligonucleotide. The specificity of this defects, RNA splicing defects, capping type of amplification depends on the fidelity of polymerase extension at a mismatch. site mutations, RNA cleavage defects, initiator codon changes, and unstable transcripts.O26) Methods to identify mer primer.Oil) Sequences would be primer and a reporter group on the ad- the precise sequence of newly arising extended in a directional manner, jacent primer gives the advantage of al- mutations rapidly have been recently using ligase to synthesize unique lowing for automation with high developed. (29,34Az7-129) LCR amplifica- dodecamer primers, until sequences on throughput, as has been elegantly tion has the potential to detect a vast both strands overlap the entire cosmid demonstrated with the analogous OLA number of mutations through a dif- clone. detection procedure.(83,8s) Such an ferent type of multiplexing format. The automated multiplex PCR/LCR detec- entire coding region of the target gene FUTURE PROSPECTS tion assay could: (1) rapidly screen would be amplified as a set of PCR Perhaps the greatest potential for LCR large populations for monogenic dis- fragments that bracket all known amplification and detection is its com- ease polymorphisms. (83,8s) (2) de- mutations. LCR amplification of patibility with a primary amplification termine HLA haplotypes for tissue several known mutations could be of genomic DNA or RNA by either typing and transplantation (117-1207 (3) tested in a single tube. Again, with a PCR (6) or 3SR.(20 One can envisage help distinguish single base deletions current signal-to-noise ratio of from 75 multiplexing the primary amplification in poly(A) or poly(G) tracts that are to greater than 500, one could of dozens of loci simultaneously, (116) not amenable to allele-specific theoretically detect the presence of one and aliquoting products into separate PCR, (121) (4) distinguish several poly- disease-carrying allele while simulta- microtiter wells. A subsequent round morphisms simutfaneously from a neously testing from 15 to 100 poten- of LCR amplification and detection single sperm to map the relative posi- tial mutations, with a fivefold higher could then distinguish a particular tions of these polymorphisms, (33,122) signal than for the combined wild-type target loci, even if it were initially and (5) help eliminate current am- signal of all those alleles tested. Im- amplified in only the attomolar range. biguities in DNA identification of indi- proving the allele-specific LCR signal- LCR amplification is compatible with viduals for forensic or paternity to-noise ratio and use of several nonradioactive, chemiluminescent, fluores- cases.(123) overlapping reporter groups (i.e., fluo- cent, or enzymatic reporter groups. The most crucial determinant in a rescent detection) could ultimately al- (8,92-94) LCR amplification creates a clinically useful disease detection assay low for rapid testing of several hun- covalent link between oligonucleo- is the signal-to-noise ratio for dis- dred polymorphic disease mutations in tides; use of a capture group on one tinguishing two alleles differing by a a given gene.

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scripts are included in the references, and any oversights or omissions are the sole responsibility of this author. This work was supported by grants from the National Institutes of Health (GM-41337-02) and the National Science Foundation (DMB-8 71435 2).

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16 PCR Methods and Applications ERRATUM TABLE 1 Ligase Chain Reaction Methods Barany, F. 1991. The ligase chain reaction in a PCR world. PCR Methods Ap- Method 1a plic. 1:5-16. Target DNA ~A,~S

Table 1 of the above titled paper in- Standard detection 1-10 attomoles (6 x 105 advertently listed an incorrect value for to 6 × 106 molecules) NAD in Method 1; the correct value is Signal-to-noise 1700 to >2000 b 1 mM, not 10 mM as stated. The cor- no target under rect version of Method 1 is reproduced standard conditions on this page. single-base mismatch 75 to >500 b under standard conditions Lowest detection 200 molecules Position of 3' base of both discriminating strands (single- nucleotide base 3' overhang) T m discrimination 64°C-68°C oligonucleotides (23- to 28-mers) T m adjacent 70°C oligonucleotides (22-mers) Amount of each 40 femtomoles oligonucleotide (0.28 ng) Volume 10 ~tl Buffer conditions 20 mM Tris-HC1, pH 7.6 c 100 mM or 150 mM KC1 10 mM MgCl 2 10 mM DTT 1 mM NAD + 1 mM EDTA

Carrier DNA to 4 ~tg of salmon sperm DNA suppress background Additional features 5' phosphate on adjacent for suppression of oligonucleotides only; target independent noncomplementary tails on background outside of oligonucleotides; single-base 3' overhang on discriminating oligonucleotides Thermostable enzyme 15 nick-closing units d Cycle conditions 94°C, 1 min 65°C, 4 rain 20 or 30 cycles

ERRATUM 94°C, 0.5 rain 65°C, 2 rain Barry, T., G. Colleran, M. Glennon, 30 or 40 cycles L.K. Dunican, and F. Gannon. 1991. The 16s/23s ribosomal spacer region as a target for DNA probes to identify eubacteria. PCR Methods Applic. 1:51-62.

Figure 4 has errors in the A1 and B1 primers. The correct primers are: A1 5 ' -AGTCGTAACAAGGTAGCCG-3 ' B1 5 '-C T/C A/G T/C TGCCAAGGCAT CCACC-3 '

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The ligase chain reaction in a PCR world.

F Barany

Genome Res. 1991 1: 5-16 Access the most recent version at doi:10.1101/gr.1.1.5

Supplemental http://genome.cshlp.org/content/suppl/2008/11/05/1.1.5.DC1 Material

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