Polymerase chain reaction techniques Richard A. Gibbs Baylor College of Medicine, Texas, USA Recent improvements to the polymerase chain reaction have produced greater priming specificity, better methods for the isolation of unknown DNA sequences, more efficient DNA recovery techniques, and new approaches to mutation detection and oligonucleotide synthesis. Current Opinion in Biotechnology 1991, 2:69-75
Introduction The in vitro amplification of DNA by the polymerase
chain reaction (PCR) is now a standard technique in molecular biology. In the first years following the original description of the method [1], an enormous number of publications appeared describing refinements of reaction parameters and variations of PCR for fragment enrichment, mutation detection, cloning, DNA sequencing, /n vitro mutagenesis and a host of other uses. Several books and reviews outlining the basic principles of PCR and the range of possible applications have also been published [2-7]. As time has passed, the frequency of appearance of reports on novel PCR applications has subsided, and PCR conditions are now often included in methods sections of manuscripts rather than cited as keywords. Nevertheless, in the period since the middle of 1989 there have been many noteworthy technical PCR papers and it is the aim of tiffs review to identify some of these and to discuss their impact. The focus is upon recent developments and so certain earlier keynote papers are omitted (although most of these may be found cited in [7]).
HOT PCR and booster PCR Successful PCRs can be performed when starting with single molecules of DNA. However, most workers find that between 10 and 1000 molecules are required to initiate reliable and reproducible amplification. Often, the failure to achieve the desired result from a limiting template is not due to an overall lack of amplification, but is the result of the generation of non-specific fragments caused by priming of the wrong template, or because of interactions between the primers themselves. Two recent reports [8,9] suggest approaches to increase PCR reliability by improving the priming specificity. The first is from Kerry Mullis the inventor of PCR, and is aptly named HOT PCR. Most unwanted extension of
oligonucleotide primers probably first occurs during the lowest temperature phase of the reaction which is usually during the initial increase of temperature prior to the first denaturation step. By simply not adding DNA polymerase until after the first high temperature plateau of PCR, a substantial increase in specificity can be achieved [8]. Alternatively, the reaction may be starved of a precursor such as a deoxyribonucleotide triphosphate until denaturation is achieved. In either case the effect is to allow only high stringency oligonucleotide binding before the polymerase activity is initiated so that the formation of non-specific PCR fragments is minimized. A comprehensive study of the subtle advantages of HOT PCR is awaited. The second suggestion for improving the reliability of amplification from limiting templates is to perform a 'booster' PCR [9]. In this procedure PCR is first performed for approximately 15 cycles using only a fraction of the normal concentration of oligonucleotide primers. Additional primers are then added and the reaction completed as usual, yielding a product free from tow molecular weight 'primer dimers' that are believed to arise from interactions between the oligonucleotides. Not only does this result in a cleaner agarose gel electrophoresis profile, it also improves the overall probability that the expected product will be recovered. Further improvements in the basic PCR protocol are possible because of a careful study of the fidelity of 7hermus acquaticus (Taq) DNA polymerase under different conditions [10oo]. The previously reported frequency of mutations induced by the enzyme can be lowered by as much as five-fold by the manipulation of pH and enzyme substrate and precursor concentrations. This high fidelity (less than one error per 100 000 bases synthesized) is unexpected because of the lack of a Taq polymerase proofreading activity. High fidelity PCR will be useful in circumstances where products are rescued by DNA cloning for sequencing and functional studies. However, the approach has the disadvantage of not using the most eft]cient reaction conditions.
Abbreviations PCR--polymerase chain reaction; Taq~ Thermus acquaticus. © Current Biology Ltd ISSN 0958-1669
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Analytical biotechnology Repeat PCR PCR has been used to amplify repetitive DNA segments with ol/gonucleotides both flanking and complementary to the repeats. Nelson and colleagues [11°°] pioneered the use of human Alu-specific PCR primers to enable the amplification and rescue of human DNA sequences in non-human DNA backgrounds. The applications of Alu PCR now reach well beyond the initial aims of detecting human-specific sequences in somatic cell hybrids and the method has been employed for cloning, mapping and walking strategies. The Alu PCR profile of amplified human sequences is reproducible, enabling DNA fragment 'fingerprints' to be identified. The large number of Alu elements (500000 per human genome) ensures a high likeliho0d of positive results. Alu-specific oligonucleotides can be paired with primers directed towards unique sequences in situations where DNA flanking a known site is to be isolated. This approach is a useful alternative to anchor PCR (described below) when working with human sequences. An added bonus of the Alu PCR system is that the ends of Alu elements often exhibit polymorphism between different individuals and can serve as useful genetic markers [12-,13]. Many Alu polymorphisms can be detected as size differences by polyacrylamide gel electrophoresis using PCR primers that flank the Alu element. Alternatively, the careful titration of an excess of a unique primer with reduced amounts of an Alu-specific oligonucleotide can give homogeneous amplification of single fragments [12.]. DNA sequence analysis may identify subtle DNA base differences between amplified Alu elements, but this procedure has not yet been widely applied because of the inconvenience of current sequencing methods. One further alternative for detection of polymorphisms in repeat DNA segments is the single-strand polymorphism detection assay [14-.], described below. Simple DNA repeats are now recognized as preferred sites for performing DNA-based linkage studies because they are frequently highly polymorphic. In addition to the variable number of tandem repeat elements, the sequences flanking short tandem repeats, such as the (CA)n motif (50 000 copies per human genome), have been extensively pursued for use as the basis of PCR tests for genetic linkage [15,16]. The value of DNA sequence information (sequence-tagged segments) as a universal language for linking different efforts to clone, map and sequence the human genome is now recognized. This information is extremely useful when combined with the repeat PCR-based methods for DNA polymorphism identification. Ideally, all sequence-tagged segments would be highly polymorphic DNA fragments, each easily assayed by a PCR-based method.
Anchor PCR Any simple, reliable method for the isolation of unknown DNA sequences that are adjacent to known sequences
would have widespread applications. Potential uses include: the rapid identification of intron-exon boundaries in complex genomes; the characterization of integration sites during recombination; the definition of DNA breakpoints involved in human disease; the isolation of variable gene sequences adjacent to constant regions; and the simplification of cloning/walking strategies. Several PCR methods have been designed for these situations, including priming from one site within a known sequence to an oligonucleotide linker that has been added by DNA ligase [17-20], and priming from a unique site to a natural or artificial homopotymeric 'tail' [21,22]. Two other methods which are useful in these situations are DNA circularization [7] and Alu PCR (see above). A choice between these protocols seems ditticult to make at first, but it can be made a little easier by examining the specific details of each method and their published performances. The DNA circularization method, for example, is a conceptually elegant protocol, but there has been a paucity of reports describing successful applications. This may reflect the technical difficulties that are intrinsic to the scheme, or it may be a consequence of the lack of attempts made by other researchers. Whichever the case, comprehensive descriptions of the application of circularization and its technical subtleties have not yet appeared. Similarly,ligation-mediated PCR has been used to study patterns of DNA methylation and for protein-binding footprints, but the method has not been described in relation to simply walking along human DNA. Two schemes can facilitate anchor PCR by the addition of a solid support capture phase after initial steps of polymerization. One approach is to add biotin to the 5' terminus of an oligonucleotide primer and to capture the PCR products using avidin-coated magnetic beads [23°°]. The alternative is to include a recognition sequence for a recombinant double-stranded DNA binding protein on the 5' end of the primer, and to capture the PCR products using the protein bound to a plastic support [24oo,25]. Either approach can function to purify desired PCR products from unwanted fragments, but the protein-binding method has the advantage of high specificity due to the preferential binding of double-stranded DNA above the single-stranded oligonucleotide. This ensures that the oligonucleotide with the protein recognition site must first be copied before it can be rescued. The PCR-based procedure that has the greatest promise for becoming the method of choice for isolation of flanking sequences is single-site PCR [26°°]. This method is the first to take advantage of the fact that great specificity can be added to a linker/ligation-based protocol by judicious choice of the orientation and sequence of linkers and primers. The method involves restriction endonuclease digestion of a DNA fragment followed by addition of a linker pair by DNA ligase (Fig. 1). The linker is joined to the DNA fragment via its 3' terminus and the ligation is facilitated by a short region of homology to a bottom 'splint' sequence. PCR is carried out between the unique priming site within the fragment and the oligonucleotide bound at the terminus. The important feature of the protocol is that the oligonucleotide that is used as a linker
Polymerase chain reaction techniques Gibbs molecule is the same as the PCR primer. In order for this molecule to fimction as a primer, the oligonucleotides added during ligation must first be copied. This can only be achieved by the extension of the unique primer, because a non-complementary overhang on the end of the splint molecule prevents base addition by the polymerase at this end of the molecule. This requirement for prior extension from the unique priming site ensures that the background observed from linker-to-linker amplification is low. The first description of this method showed strong evidence that specific amplification of unique human sequences was possible. It may be predicted that single-site PCR, perhaps in conjunction with one of the solid support related-methods described above, is an approach that will be used with great success. A similar protocol uses a 'bubble primer' to avoid the priming of all ligated fragments and has been applied to the problem of isolation of the termini of inserts cloned into yeast artificial chromosomes [27].
propagation of recombinant DNA libraries, but with the advantages of greater speed and efficiency. The process of amplification of a mixed population of DNA molecules is termed here 'regenerative PCR'. Recent successful applications of regenerative PCR include the improvement of subtractive cDNA screening [28], rescue of microdissected human chromosomes [29o°], isolation of fragments of human or synthetic DNA that bind to specific ligands [30..,31oo], and propagation of cloned cDNA libraries for the generation of antibodies [32,33]. The subtractive screening and chromosome microdissection strategies are recognizable as methods that have previously functioned with reduced efficiency without PCR. In contrast, the addition of PCR to the ligand-binding studies and to the antibody cDNA library construction has generated entirely new approaches to the selection of individual DNA fragments. The diversity of these studies dramatically illustrates the wide-ranging impact of PC1L An important qualification to the universal applicability of regenerative PCR is that unequal amplification of different fragments may occur. Factors such as different lengths between priming sites, variations in DNA sequence composition or changes in initial abundance could each skew the representation of different DNA sequences in an amplified mixture. No single study has rigorously addressed these points, although several of the reports cited here have discussed specific controls that, under restricted conditions, allow approximately equal amplification to
Regenerative PCR Many potentially powerful techniques in molecular biology are limited by the low efficiency of DNA recovery during a single step. PCR can be integrated into these methods following ligation of oligonucleotides to the ends of DNA fragments and the use of those sites to prime simultaneous synthesis of all DNA in the mixture. In this manner it is possible to amplify in vitro entire populations of molecules in a fashion analogous to the construction and
occur.
(a)
3'
5!
5'3' 35;' (b)
I
~'I
~Ligation
V
(c)
x _~
3' 3'
5'
3'
3I
5'
fig. 1. Single-site polymerase chain reaction (PCR) for the amplification of fragments of unknown sequence adjacent to known sequence. (a) The DNA is first cut with a restriction endonuclease that recognizes DNA outside of the known sequence. (b) An oligonucleotide linker is added to the end of each newly created fragment by ligation. The oligonucleotide linker (upper strand) is joined by the 3' terminus and is held in place by a short oligonucleotide splint (lower strand). The splint molecule is not completely complementary to the opposite strand, which inhibits subsequent extension by Taq polymerase. (c) The ligation products are then amplified by oligonucleotides that bind either to the linker site (x) or to the unique site (y). In the first PCR cycle the oligonucleotide that binds to the unique site will be extended to generate a copy of the linker region. In subsequent cycles, the primer directed toward the linker site will be able to bind to produce the amplified DNA fragment. The specificity of the overall reaction relies upon the necessity of the linker region to be copied before the primer (x) can function.
71
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Analytical biotechnoloBy Quantitative PCR
ods add to the previous array of procedures, and have unique advantages in certain circumstances.
PCR amplifications generally occur with reproducible efficiencies, and this can allow quantitative estimates of DNA template concentrations. The requirements for good quantitative PCR assays are the availability of internal standards, a willingness to construct the proper standard curves, and a statistical analysis of the accuracy of the results. In the case of DNA templates this is relatively easy to achieve, although each system needs to be carefully and rigorously standardized. Internal reference fragments that are amplified by the same primers as test fragments have been used to overcome possible differences in amplification efficiency with different oligonucleotides [34]. However, most workers have relied on unrelated primer sets to generate standard curves.
The fluorescence-detection procedure [37] is particularly striking as the end-product is a solution that appears as a different color under ultra-violet illumination, depending upon the genotype tested. Two or more dye-labelled oligonucleotides are included in the reaction and incorporated during PCR depending upon the presence or absence of the matching template. Single DNA base differences can be identified by taldng advantage of the competition between perfectly matched oligonucleotides and mismatched oligonucleotides. In this way, the assay may prove useful for screening to detect common human disease alleles.
Accurate RNA quantitation via PCR is more difficult to achieve than DNA measurement because the efficiency of reverse transcription may vary from tube to tube, and may be affected by the initial abundance of the target. One solution for controlling the varying efficiency of individual reverse wan~riptase reactions is the use of an internal synthetic RNA standard. Unfortunately, RNA that is enzymatically synthesized in vitro is often contaminated with DNA that cannot easily be removed, even by extensive DNAase treatment. Chemical synthesis of long RNA molecules is not yet practicable. Wang eta/. [35] have used oligo(dT) to purify in vitro synthesized RNA standards that contain poly(A) tracts to achieve an accurate quantitation of the expression of several different messengers. A novel use of quantitative PCR following reverse transcription is in the examination of the relative abundance of processed versus unprocessed heteronuclear RNA. Synthesis of appropriate oligonucleotides allows the simultaneous amplification of short (spliced) RNA fragments as well as longer (unspliced) fragments containing parts of adjacent introns [36]. Alterations to the ratio of the two species can, therefore, be related to the rate of initiation of new synthesis following a period of no synthesis. This statistic is similar to the information generated by nuclear run-on assays, but is distinctive in being very much easier to generate.
Mutation detection by PCR One of the first applications of PCR was to simplify the isolation and characterization of mutant DNA fragments. Consequently, many of the mutation-detection techniques that have been combined with PCR have been reviewed already [2-7]. These techniques include chemical mismatch cleavage, denaturing gradient gel electrophoresis, forward and reverse allele-specific hybridization, competitive oligonucleotide priming and allele-specific priming (each cited in [7]). More recently, PCR has been combined with fluorescence for detection of mutations [37] and used in a single-stranded conformation polymorphism detection assay [14°°]. These two meth-
The applications of a single-stranded conformation polymorphism assay for detection of variations in repeated DNA are mentioned above. For detection, the radiolabeled products of PCR amplification are denatured and analyzed by native polyacrylamide gel electrophoresis [14 °°]. Single DNA base substitutions alter the electrophoretic migration of the single-stranded molecules, presumably as a consequence of alterations in the internal base-pairing. The single-stranded conformation polymorphism detection assay is very simple to perform and, therefore, is likely to be favored for the rapid screening of PCR products for polymorphisms. It is not yet known if every possible single DNA base difference can be detected by this procedure and, because of this, it cannot be used in situations where 100% ascertainment is essential.
Sequencing PCR products The sequences of PCR products can be determined either directly, or after cloning of the products into suitable vectors. The advantages of the direct sequence analysis have been discussed extensively elsewhere [2-7,38] and the procedure has been carried out by many investigators. Despite this, direct sequencing is often found to be difficult or unreliable and a protocol that is universally regarded as superior to any other has yet to be identified. In our studies of mutations leading to human genetic diseases, we have developed reliable protocols for either radioactive or fluorescent automated DNA sequencing that are sufficiently sensitive to enable detection of heterozygous single DNA base substitutions [38,39]. Other investigators have used slightly different procedures with similar success and, in general, it seems that the precise factors that will lead to consistently good PCR-direct sequencing are not yet understood. Most of the procedures used are based on the concept of asymmetric PCR [40], or some other method for the generation of a singlestranded template, followed by conventional dideoxynucleotide sequencing. One advance on previous methods for the generation of single strands for PCR sequencing is based upon the use of solid supports. A biotin residue is first added to the 5' terminus of one oligonucleotide and then the primer
Polymerasechain reaction techniquesGibbs 73 incorporated by PCR into the DNA fragment to be analyzed. The biotinylated strand can be conveniently captured using an avidin-coated magnetic bead, so that it may be purified from the complementary DNA prior to the sequencing [23--,39]. Solid-support sequencing has also been demonstrated using an avidin-bound column, but the beads offer greater convenience and the possibility for automation [41]. In our studies, the ability to thoroughly wash DNA bound to solid supports in order to remove salt and unwanted PCR products has improved the quality and reliability of subsequent fluorescent-automated DNA sequences.
PCR from microorganisms It is very likely that as the necessary DNA sequence information becomes ~ailable PCR will continue to displace more tedious assays for the presence of microorganisms. There have been innumerable examples of PCR-based microbe detection, but few of these represent major advances in technology. One exception is the antigen-capture polymerase chain reaction method, which has elegantly combined the specificity of the antibody-recognition of type A hepatitis virus with the sensitivity of PCR [42]. Antibodies are attached to a microtitre plate well which is then exposed to samples containing viral particles. The captured virus is retained during a washing step and then the nucleic acid of the viral genome is amplified by specific oligonucleotide primers. Finally, the PCR product is detected by gel electrophoresis. The combination of two independent methods for specific target selection will no doubt be useful for the reliable detection of many other microorganisms. The presence of conserved rRNA flanking variable sequences in most bacteria is allowing the identification of species-specific oligonucleotide priming sites [43]. First, rRNA-specific primers are used to amplify segments of DNA that contain the variable regions. The variable DNA is then sequenced and new oligonucleotide primers are generated which can be used to specifically amplify fragments from only that species. Alternatively, conserved primers can be used routinely for amplification, and another method employed to distinguish the variable segments.
PCR of synthetic oligonucleotides The use of PCR to improve the synthesis of long oligonucleotides has been elegantly demonstrated by Barnett and Erfle [44.]. The crude products of synthesis of a 234 base sequence were amplified by short PCR primers complementary to either end of the molecule. The successful amplification was monitored by agarose gel electrophoresis and the products cloned and sequenced to verify that no errors had been introduced by either chemical or enzymatic steps in the procedure. When compared with more conventional approaches of chemically synthesiz-
ing both strands of the new sequence, or of constructing partially overlapping oligonucleotides that are then copied once enzymatically, the new method is cheaper, faster and more reliable. The approach is an extremely useful addition to previous methods of 'gene synthesis' and will no doubt encourage many individuals to attempt DNA constructions that would otherwise have been too costly or time-consuming.
Contamination Finally, Sarker and Sommer [45 .°] have established the first method that does not require the usual extensive precautions against contamination of PCR by exogenous DNA fragments. Modest doses of short wavelength ultraviolet light prevent contaminating double-stranded flagments from functioning as PCR templates, but do not interfere with the ability of single-stranded, short oligonucleotides to prime the desired fragments efficiently. It can be anticipated that ultraviolet 'sterilization' of the environment where PCR mixes are constructed will become a standard procedure in laboratories where contamination is a problem.
Conclusion Innovative PCR-based methods have been developed throughout the previous year, thus expanding the list of applicaions that have been described since 1985. This trend will probably continue as the procedure is constantly adapted for use in situations where analysis or manipulation of DNA is limited by the amount or relative abundance of desired DNA fragments.
References and recommended reading Papers of specialinterest,publishedwithin the annualperiod of review, have been highlightedas: • of interest •. of outstanding interest 1.
MULLIS KB, FALOONAF: SpecificSynthesisof DNA in vitro via a Polymerase-CatalyzedChain Reaction. Methods Enzymol 1987, 155:335-350.
2.
ERLICHHA (ed): PCR Technology: Princ~oles and applicatz'ons for DNA ampUflcatio~ New York: Stockton Press, 1990.
3.
MuuasKB: The Unusual Origins of the Polymerase Chain Reaction. Sci Am 1990, 263:56-65.
4.
INNISMA, GELFANDDH, SNINSKYJJ, WHITETJ (eds): PCR Protocols. A guide to methods and a/~l#_.att~ns San Diego: Academic Press, Inc. 1990.
5.
ERLICH HA, GIBBS RA, KAZAZIANJNRI-IH(eds): The Po/ymerase Chain Reaction New York: Cold Spring Harbor Press, 1989.
6.
WHrrETJ, ARNHEtMN, ERLICHHA: The Polymerase Chain Reaction. Trends Genet 1989, 5:185--188.
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Analytical
biotechnology
7.
Gram RA: DNA Amplification by the Polymerase Chain Reaction. A n a / t ~ e m 1990, 62:1202-1214.
8.
MULLISIG Targeted Amplification of DNA by the Polymerase Chain Reaction. Annals Cloem Clinique 1990, 20.
9.
RUANOG, FENTON W, KIDD KK: Biphasic Amplification of Very Dilute DNA Samples via 'Booster' PCR. Nucleic Acids Res 1989, 17:5407.
10.
ECKERTKA, KUNKEL TA: High Fidelity DNA Synthesis by
•.
the T h e r m u s Aquatloats DNA Polymerase. Nucleic Acids Res
1990, 18:3739-3744. A thorough study of factors which are relevant to PCR conditions and which influence the ram of mutations induced by Taq DNA polymerase. 11. tto
NELSONn l , LEDBETI'ERS~ CORBO I~ VICTORIAM, RAMARIZ-SOLIS R, WEBSTERTD, LEDBZITERDH, CASKEYCT: AIu Polymerase Chain Reaction: A Method for Rapid Isolation of Human Specific Sequences from Complex DNA Sources. Proc Natl Acad Sci USA 1989, 85:6686-6690. The first description of the use of Alu-specific PCR primers for isolation of human DNA sequences. Alu PCR has since become an important tool in human molecular genetics. 12. •
ECONOMOUEP, BERGENA, WARRENAC, ANTONARAKISSE: The Polydeoxyadenylate Tract of Alu Repetitive Elements is Polymorphic in the Human Genome. Proc Natl Acad SCi USA 1989, 85:6686-6690. This paper defines the conditions for reliable amplification of fragments defined by Alu-specific primers and oligonudeotides that bind to unique sites. It also reports valuable biological observations.
Magnetic Beads as a Solid Support. Nucleic Acids Res 1989, 17:4937-4946. Magnetic beads are used as solid supports for PCR fragments by the introduction of biotin residues at the 5' end of oligonudeotide primers. The ease of manipulation of the bound fragments makes this procedure very useful for many applications. 24. •s
KEMPDJ, SM1TH DB, FOOTE SJ, SAMARASN, PETERSON MG: Colorimetric Detection of Specific DNA Segments Amplified by Polymerase Chain Reactions. Proc Natl Aca~ Sci USA 1989, 86:2423-2427. The combination of a double-stranded DNA binding-protein and PCR are used here to specifically capture and detect amplified DNA fragments. This general approach is applicable to microtitre plate formats and, therefore, may be favored for large scale screening. 25.
Izw AM, KEMP DJ: Isolating DNA Segments from Cloned Libraries Without Screening by Afl~inity Selection of PCR Products. Nucleic Acids Res 1989, 17:5859-5860.
26. •.
ROUX KH, DI-IANARMANP: A Strategy for Single Site PCR Amplification of dsDN& Priming Digested Cloned or Genomic DNA from an Anchor-Modified Restriction Site and a Short Internal Sequence. B/otechm~_~ 1990, 8:48-57. Avery clever method for performing anchor PCK This is the first report that makes use of prior internal priming in a fragment as a prerequisite for end priming, so as to give a highly specific anchor PCR strategy. 27.
RILEYJ, BUTLERR, OGILVlE D, FINNIEARR, JENNER D, POWELL S, ANAND R, SMITHJC, MARKHAMAF: A Novel, Rapid Method for the Isolation of Terminal Sequences from Yeast Artificial Chromosome (YAC) Clones. Nucleic Acids Res 1990, 18:2887-2890.
EPSTE~N, NAHOR O, SILVERJ: The 3' Ends of Alu Repeats are Highly Polymorphic. Nuc/e/c Acids Res 1990, 18:4634.
28.
ORITAM, SUZU~ Y, SEKIYA T, HAYASHI K: Rapid and SCnsitive Detection of Point Mutations and DNA Polymorphisms Using the Polymerase Chain Reaction. Genom#.s 1989, 5:874-879. The single-strand conformation polymorphism detection assay is technically the most simple method for scanning for mutations that has yet been described.
TIMBLIN C, BATIXY J, KUEHL WM: Application for PCR Technology to Subtractive cDNA Cloning: Identification of Genes Expressed Specifically in Murine Plasmacytoma Cells. Nude/c Ac/ds Res 1990, 18:1587-1593.
29. •.
13. 14. e.
15.
~(rEBERJI~ Informativeness of Human (dCMA)n(dG-dT)n Polymorphisms. Genom~s 1990, 7:524-530.
16.
AU S, WAL~CE RB: Intrinsic Polymorphism of Variable Number of Tandem Repeat Loci in the Human Genome. Nuc/e~ Act~ Res 1988, 16:8487-43496.
17.
MEULLERPR, WOLDE B: I n v i v o Footprinting of a Muscle Specific Enhancer by Ltgation Mediated PCR. Science 1989, 246:78O-786.
18.
PFEIFERGP, STEIGERWALDSD, MUELLERPR, WOLD B, R1GGSAD: Genomic Sequencing and Methylation Analysis by Ligation Mediated PCR. Science 1989, 246:780-786.
19.
ROSENTHALA, JONES DS: Genomic Walking and Sequencing by Olig~Cassette Mediated Polymerase Chain Reaction. Nucleic Acids Res 1990, 18:3095-3096.
20.
FORS ~ SAAVEDRARA, HOOD 1.. Cloning of the Shark PO Promoter Using a Genomic Walking Technique Based on the Polymerase Chain Reaction. Nucleic Acids Res 1990, 18:2793-2799.
21.
LOH EY, ELLIOTJF, CWERLAS, LANIERLL, DAVISMM: Polymerase Chain Reaction with Single Sided Specificity: Analysis of TCell Receptor Delta Chain. Sc/ence 1989, 243:217-220.
22.
FROHMANMA, DUSH MK, MARTINGR: Rapid Production of Full Lensth cDNAs from Rare Transcripts Using Single Gene Specific Oiigonucleotide Primer. Proc Nail Acad Sci USA 1988, 85:8998--9002.
23. ..
HULTMANT, STAHLL S, HORNES E, UHI£N M: Direct Solid Phase Sequencing of Genomic and Plasmid DNA Using
IX/DECKEHJ, SENGERG, CLAUSSENU, HOSSTHEMKEB: Cloning Defined Regions of the Human Genome by Microdissection of Banded Chromosomes and Enzymatic Amplification. Nature 1989, 338:348-350. Although microdissection of chromosomes is technically very difficult, PCR makes possible the efficient recovery of the minute amounts of material generated. 30. •.
KINZER~ , VOGELSTEINB: Whole Genome PCPc Application to the Identification of Sequences Bound by Gene Regulatory Proteins. Nucleic Acids Res 1989, 17:3645-3653. PCR can be used to help overcome the difficulties previously associated with screening specific DNA fragments for protein binding. 31. ..
TUERKC, GOLD I~ Systematic Evolution of Ltgands by Exponenttal Enrichment: RNA l.iwands to Bacteriophage T4 DNA Polymerase. Science 1990, 249:505-510. A report of a new approach to the study of DNA ligand interactions. 32.
HUSEX,VD, SASTRY lo IVERSON SA, KANG AS, ALTING-MEES M, BURTON DR, BENKOVICSJ, LERNERRA: Generation of a Large Combinational Library of the Immunoglobulin Repertoire in Phage Lambda. Science 1989, 246:1275-1281.
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CHAUDHARYVK, BATRA JK, GALLD MG, WIIL~GHAM MC, FrlXGERALDDJ, PASTANI: A Rapid Method of Cloning Functional Variable-Region Antibody Genes in Escherichia Coil as Single-Chain lmmunotoxins. Proc N a a Acad Sci USA 1990, 87:1066-1070.
34.
GIIJJIANDG, PERR1N S, BLANCHARDK, BUNN HF: Analysis of Cytokine mRNA and DNA: Detection and Quantitation by Competitive Polymerase Chain Reaction. Proc N a a Acad SCi USA 1990, 87:2725-2729.
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WANGAM, DOYLE MV, MASK DF: Quantitation of mRNA by the Polymerase Chain Reaction. Proc N a a Acad SCi USA 1989, 86:9717-9721.
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LIPSONKE, BASERGAR: Transcriptional Activity of the Human Thymidine Kinase Gene Determined by a Method using the
Polymerasechain Polymerase Chain Reaction and an Intron-Specific Probe. Proc Natl Acad Sci USA 1989, 86:9774-9777.
Polymerase Chain Reaction Method. Proc Natl Acad S¢i USA 1990, 87:2867-2871.
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CHEHABFF, KAN YW: Detection of Specific DNA Sequences by Fluorescence Amplification: A Color Complementation Assay. Proc N a a Acad Sci USA 1989, 86:9178-9182.
43.
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GraBSRA, NGUYENPN, CASKEYCT: Direct DNA Sequencing of Complementary DNA Amplified by the Polymerase Chain Reaction. In Methods in Molecular BiologF ec~ted by Mathew C. London: Humana press, 1991, in press.
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GIBBSRA, NGUYEN PN, EDWARDS A, CIVITELLO AB, CASKEY CT: Multiplex DNA Deletion Detection and Exon Sequencing of the Hypoxanthine P h o a p h o ~ t r a n s f e r a s e Gene in Lesch-Nyhan Families. Genom/cs 1990, 7:235-244.
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GYLLENSTENUB, ERLICH HA: Generation of Single-Stranded DNA by the PolymeraseChain Reaction and its Application to Direct Sequencing of the HLA-DQA Locus. Proc Natl Acad Sci USA 1988, 85:7652-7656.
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MITCHELLLG, MERmL CR: Affinity Generation of SingleStranded DNA for Dideoxy Sequencing Following the Polymerase Chain Reaction. Anal B~cJ0em 1989, 178:239-242.
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JANSENRW, SIEGL G, LEMONSM: Molecular Epidemiology of Human Hepatitis A Virus Defined by an Antigen Capture
reaction techniques Gibbs
BOTrGER EC: Rapid Determination of Bacterial Ribosomal RNA Sequences by Direct Sequencing of Enzymatically Amplified DNA. FEMS Microbiol Lett 1989, 53:171-176.
BARNETtRW, ERFLE H: Rapid Generation of DNA Fragments by PCR Amplification of Crude, Synthetic Oligonucleotides. Nucleic Acids Res 1990, 18:3094. Efficient recovery of synthetic DNA longer than 50 bases is generally very diflqcult but here, PCR is used to generate the desitx~d fragment from the products of synthesis of a 234 base sequence. This process will considerably reduce the cost of 'gene synthesis' projects. 45.
SARKERG, SOMMERSS: Shedding Light on PCR Contaminatiotx Nature 1990, 343:27. ~ e simplest way yet described for getting rid of unwanted PCR contamination.
RA Gibbs, Institute for Molecular Genetics, Baytor College of Medicine, Texas Medical Center, Houston, Texas 77030, US&
75
Introduction The in vitro amplification of DNA by the polymerase
chain reaction (PCR) is now a standard technique in molecular biology. In the first years following the original description of the method [1], an enormous number of publications appeared describing refinements of reaction parameters and variations of PCR for fragment enrichment, mutation detection, cloning, DNA sequencing, /n vitro mutagenesis and a host of other uses. Several books and reviews outlining the basic principles of PCR and the range of possible applications have also been published [2-7]. As time has passed, the frequency of appearance of reports on novel PCR applications has subsided, and PCR conditions are now often included in methods sections of manuscripts rather than cited as keywords. Nevertheless, in the period since the middle of 1989 there have been many noteworthy technical PCR papers and it is the aim of tiffs review to identify some of these and to discuss their impact. The focus is upon recent developments and so certain earlier keynote papers are omitted (although most of these may be found cited in [7]).
HOT PCR and booster PCR Successful PCRs can be performed when starting with single molecules of DNA. However, most workers find that between 10 and 1000 molecules are required to initiate reliable and reproducible amplification. Often, the failure to achieve the desired result from a limiting template is not due to an overall lack of amplification, but is the result of the generation of non-specific fragments caused by priming of the wrong template, or because of interactions between the primers themselves. Two recent reports [8,9] suggest approaches to increase PCR reliability by improving the priming specificity. The first is from Kerry Mullis the inventor of PCR, and is aptly named HOT PCR. Most unwanted extension of
oligonucleotide primers probably first occurs during the lowest temperature phase of the reaction which is usually during the initial increase of temperature prior to the first denaturation step. By simply not adding DNA polymerase until after the first high temperature plateau of PCR, a substantial increase in specificity can be achieved [8]. Alternatively, the reaction may be starved of a precursor such as a deoxyribonucleotide triphosphate until denaturation is achieved. In either case the effect is to allow only high stringency oligonucleotide binding before the polymerase activity is initiated so that the formation of non-specific PCR fragments is minimized. A comprehensive study of the subtle advantages of HOT PCR is awaited. The second suggestion for improving the reliability of amplification from limiting templates is to perform a 'booster' PCR [9]. In this procedure PCR is first performed for approximately 15 cycles using only a fraction of the normal concentration of oligonucleotide primers. Additional primers are then added and the reaction completed as usual, yielding a product free from tow molecular weight 'primer dimers' that are believed to arise from interactions between the oligonucleotides. Not only does this result in a cleaner agarose gel electrophoresis profile, it also improves the overall probability that the expected product will be recovered. Further improvements in the basic PCR protocol are possible because of a careful study of the fidelity of 7hermus acquaticus (Taq) DNA polymerase under different conditions [10oo]. The previously reported frequency of mutations induced by the enzyme can be lowered by as much as five-fold by the manipulation of pH and enzyme substrate and precursor concentrations. This high fidelity (less than one error per 100 000 bases synthesized) is unexpected because of the lack of a Taq polymerase proofreading activity. High fidelity PCR will be useful in circumstances where products are rescued by DNA cloning for sequencing and functional studies. However, the approach has the disadvantage of not using the most eft]cient reaction conditions.
Abbreviations PCR--polymerase chain reaction; Taq~ Thermus acquaticus. © Current Biology Ltd ISSN 0958-1669
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Analytical biotechnology Repeat PCR PCR has been used to amplify repetitive DNA segments with ol/gonucleotides both flanking and complementary to the repeats. Nelson and colleagues [11°°] pioneered the use of human Alu-specific PCR primers to enable the amplification and rescue of human DNA sequences in non-human DNA backgrounds. The applications of Alu PCR now reach well beyond the initial aims of detecting human-specific sequences in somatic cell hybrids and the method has been employed for cloning, mapping and walking strategies. The Alu PCR profile of amplified human sequences is reproducible, enabling DNA fragment 'fingerprints' to be identified. The large number of Alu elements (500000 per human genome) ensures a high likeliho0d of positive results. Alu-specific oligonucleotides can be paired with primers directed towards unique sequences in situations where DNA flanking a known site is to be isolated. This approach is a useful alternative to anchor PCR (described below) when working with human sequences. An added bonus of the Alu PCR system is that the ends of Alu elements often exhibit polymorphism between different individuals and can serve as useful genetic markers [12-,13]. Many Alu polymorphisms can be detected as size differences by polyacrylamide gel electrophoresis using PCR primers that flank the Alu element. Alternatively, the careful titration of an excess of a unique primer with reduced amounts of an Alu-specific oligonucleotide can give homogeneous amplification of single fragments [12.]. DNA sequence analysis may identify subtle DNA base differences between amplified Alu elements, but this procedure has not yet been widely applied because of the inconvenience of current sequencing methods. One further alternative for detection of polymorphisms in repeat DNA segments is the single-strand polymorphism detection assay [14-.], described below. Simple DNA repeats are now recognized as preferred sites for performing DNA-based linkage studies because they are frequently highly polymorphic. In addition to the variable number of tandem repeat elements, the sequences flanking short tandem repeats, such as the (CA)n motif (50 000 copies per human genome), have been extensively pursued for use as the basis of PCR tests for genetic linkage [15,16]. The value of DNA sequence information (sequence-tagged segments) as a universal language for linking different efforts to clone, map and sequence the human genome is now recognized. This information is extremely useful when combined with the repeat PCR-based methods for DNA polymorphism identification. Ideally, all sequence-tagged segments would be highly polymorphic DNA fragments, each easily assayed by a PCR-based method.
Anchor PCR Any simple, reliable method for the isolation of unknown DNA sequences that are adjacent to known sequences
would have widespread applications. Potential uses include: the rapid identification of intron-exon boundaries in complex genomes; the characterization of integration sites during recombination; the definition of DNA breakpoints involved in human disease; the isolation of variable gene sequences adjacent to constant regions; and the simplification of cloning/walking strategies. Several PCR methods have been designed for these situations, including priming from one site within a known sequence to an oligonucleotide linker that has been added by DNA ligase [17-20], and priming from a unique site to a natural or artificial homopotymeric 'tail' [21,22]. Two other methods which are useful in these situations are DNA circularization [7] and Alu PCR (see above). A choice between these protocols seems ditticult to make at first, but it can be made a little easier by examining the specific details of each method and their published performances. The DNA circularization method, for example, is a conceptually elegant protocol, but there has been a paucity of reports describing successful applications. This may reflect the technical difficulties that are intrinsic to the scheme, or it may be a consequence of the lack of attempts made by other researchers. Whichever the case, comprehensive descriptions of the application of circularization and its technical subtleties have not yet appeared. Similarly,ligation-mediated PCR has been used to study patterns of DNA methylation and for protein-binding footprints, but the method has not been described in relation to simply walking along human DNA. Two schemes can facilitate anchor PCR by the addition of a solid support capture phase after initial steps of polymerization. One approach is to add biotin to the 5' terminus of an oligonucleotide primer and to capture the PCR products using avidin-coated magnetic beads [23°°]. The alternative is to include a recognition sequence for a recombinant double-stranded DNA binding protein on the 5' end of the primer, and to capture the PCR products using the protein bound to a plastic support [24oo,25]. Either approach can function to purify desired PCR products from unwanted fragments, but the protein-binding method has the advantage of high specificity due to the preferential binding of double-stranded DNA above the single-stranded oligonucleotide. This ensures that the oligonucleotide with the protein recognition site must first be copied before it can be rescued. The PCR-based procedure that has the greatest promise for becoming the method of choice for isolation of flanking sequences is single-site PCR [26°°]. This method is the first to take advantage of the fact that great specificity can be added to a linker/ligation-based protocol by judicious choice of the orientation and sequence of linkers and primers. The method involves restriction endonuclease digestion of a DNA fragment followed by addition of a linker pair by DNA ligase (Fig. 1). The linker is joined to the DNA fragment via its 3' terminus and the ligation is facilitated by a short region of homology to a bottom 'splint' sequence. PCR is carried out between the unique priming site within the fragment and the oligonucleotide bound at the terminus. The important feature of the protocol is that the oligonucleotide that is used as a linker
Polymerase chain reaction techniques Gibbs molecule is the same as the PCR primer. In order for this molecule to fimction as a primer, the oligonucleotides added during ligation must first be copied. This can only be achieved by the extension of the unique primer, because a non-complementary overhang on the end of the splint molecule prevents base addition by the polymerase at this end of the molecule. This requirement for prior extension from the unique priming site ensures that the background observed from linker-to-linker amplification is low. The first description of this method showed strong evidence that specific amplification of unique human sequences was possible. It may be predicted that single-site PCR, perhaps in conjunction with one of the solid support related-methods described above, is an approach that will be used with great success. A similar protocol uses a 'bubble primer' to avoid the priming of all ligated fragments and has been applied to the problem of isolation of the termini of inserts cloned into yeast artificial chromosomes [27].
propagation of recombinant DNA libraries, but with the advantages of greater speed and efficiency. The process of amplification of a mixed population of DNA molecules is termed here 'regenerative PCR'. Recent successful applications of regenerative PCR include the improvement of subtractive cDNA screening [28], rescue of microdissected human chromosomes [29o°], isolation of fragments of human or synthetic DNA that bind to specific ligands [30..,31oo], and propagation of cloned cDNA libraries for the generation of antibodies [32,33]. The subtractive screening and chromosome microdissection strategies are recognizable as methods that have previously functioned with reduced efficiency without PCR. In contrast, the addition of PCR to the ligand-binding studies and to the antibody cDNA library construction has generated entirely new approaches to the selection of individual DNA fragments. The diversity of these studies dramatically illustrates the wide-ranging impact of PC1L An important qualification to the universal applicability of regenerative PCR is that unequal amplification of different fragments may occur. Factors such as different lengths between priming sites, variations in DNA sequence composition or changes in initial abundance could each skew the representation of different DNA sequences in an amplified mixture. No single study has rigorously addressed these points, although several of the reports cited here have discussed specific controls that, under restricted conditions, allow approximately equal amplification to
Regenerative PCR Many potentially powerful techniques in molecular biology are limited by the low efficiency of DNA recovery during a single step. PCR can be integrated into these methods following ligation of oligonucleotides to the ends of DNA fragments and the use of those sites to prime simultaneous synthesis of all DNA in the mixture. In this manner it is possible to amplify in vitro entire populations of molecules in a fashion analogous to the construction and
occur.
(a)
3'
5!
5'3' 35;' (b)
I
~'I
~Ligation
V
(c)
x _~
3' 3'
5'
3'
3I
5'
fig. 1. Single-site polymerase chain reaction (PCR) for the amplification of fragments of unknown sequence adjacent to known sequence. (a) The DNA is first cut with a restriction endonuclease that recognizes DNA outside of the known sequence. (b) An oligonucleotide linker is added to the end of each newly created fragment by ligation. The oligonucleotide linker (upper strand) is joined by the 3' terminus and is held in place by a short oligonucleotide splint (lower strand). The splint molecule is not completely complementary to the opposite strand, which inhibits subsequent extension by Taq polymerase. (c) The ligation products are then amplified by oligonucleotides that bind either to the linker site (x) or to the unique site (y). In the first PCR cycle the oligonucleotide that binds to the unique site will be extended to generate a copy of the linker region. In subsequent cycles, the primer directed toward the linker site will be able to bind to produce the amplified DNA fragment. The specificity of the overall reaction relies upon the necessity of the linker region to be copied before the primer (x) can function.
71
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Analytical biotechnoloBy Quantitative PCR
ods add to the previous array of procedures, and have unique advantages in certain circumstances.
PCR amplifications generally occur with reproducible efficiencies, and this can allow quantitative estimates of DNA template concentrations. The requirements for good quantitative PCR assays are the availability of internal standards, a willingness to construct the proper standard curves, and a statistical analysis of the accuracy of the results. In the case of DNA templates this is relatively easy to achieve, although each system needs to be carefully and rigorously standardized. Internal reference fragments that are amplified by the same primers as test fragments have been used to overcome possible differences in amplification efficiency with different oligonucleotides [34]. However, most workers have relied on unrelated primer sets to generate standard curves.
The fluorescence-detection procedure [37] is particularly striking as the end-product is a solution that appears as a different color under ultra-violet illumination, depending upon the genotype tested. Two or more dye-labelled oligonucleotides are included in the reaction and incorporated during PCR depending upon the presence or absence of the matching template. Single DNA base differences can be identified by taldng advantage of the competition between perfectly matched oligonucleotides and mismatched oligonucleotides. In this way, the assay may prove useful for screening to detect common human disease alleles.
Accurate RNA quantitation via PCR is more difficult to achieve than DNA measurement because the efficiency of reverse transcription may vary from tube to tube, and may be affected by the initial abundance of the target. One solution for controlling the varying efficiency of individual reverse wan~riptase reactions is the use of an internal synthetic RNA standard. Unfortunately, RNA that is enzymatically synthesized in vitro is often contaminated with DNA that cannot easily be removed, even by extensive DNAase treatment. Chemical synthesis of long RNA molecules is not yet practicable. Wang eta/. [35] have used oligo(dT) to purify in vitro synthesized RNA standards that contain poly(A) tracts to achieve an accurate quantitation of the expression of several different messengers. A novel use of quantitative PCR following reverse transcription is in the examination of the relative abundance of processed versus unprocessed heteronuclear RNA. Synthesis of appropriate oligonucleotides allows the simultaneous amplification of short (spliced) RNA fragments as well as longer (unspliced) fragments containing parts of adjacent introns [36]. Alterations to the ratio of the two species can, therefore, be related to the rate of initiation of new synthesis following a period of no synthesis. This statistic is similar to the information generated by nuclear run-on assays, but is distinctive in being very much easier to generate.
Mutation detection by PCR One of the first applications of PCR was to simplify the isolation and characterization of mutant DNA fragments. Consequently, many of the mutation-detection techniques that have been combined with PCR have been reviewed already [2-7]. These techniques include chemical mismatch cleavage, denaturing gradient gel electrophoresis, forward and reverse allele-specific hybridization, competitive oligonucleotide priming and allele-specific priming (each cited in [7]). More recently, PCR has been combined with fluorescence for detection of mutations [37] and used in a single-stranded conformation polymorphism detection assay [14°°]. These two meth-
The applications of a single-stranded conformation polymorphism assay for detection of variations in repeated DNA are mentioned above. For detection, the radiolabeled products of PCR amplification are denatured and analyzed by native polyacrylamide gel electrophoresis [14 °°]. Single DNA base substitutions alter the electrophoretic migration of the single-stranded molecules, presumably as a consequence of alterations in the internal base-pairing. The single-stranded conformation polymorphism detection assay is very simple to perform and, therefore, is likely to be favored for the rapid screening of PCR products for polymorphisms. It is not yet known if every possible single DNA base difference can be detected by this procedure and, because of this, it cannot be used in situations where 100% ascertainment is essential.
Sequencing PCR products The sequences of PCR products can be determined either directly, or after cloning of the products into suitable vectors. The advantages of the direct sequence analysis have been discussed extensively elsewhere [2-7,38] and the procedure has been carried out by many investigators. Despite this, direct sequencing is often found to be difficult or unreliable and a protocol that is universally regarded as superior to any other has yet to be identified. In our studies of mutations leading to human genetic diseases, we have developed reliable protocols for either radioactive or fluorescent automated DNA sequencing that are sufficiently sensitive to enable detection of heterozygous single DNA base substitutions [38,39]. Other investigators have used slightly different procedures with similar success and, in general, it seems that the precise factors that will lead to consistently good PCR-direct sequencing are not yet understood. Most of the procedures used are based on the concept of asymmetric PCR [40], or some other method for the generation of a singlestranded template, followed by conventional dideoxynucleotide sequencing. One advance on previous methods for the generation of single strands for PCR sequencing is based upon the use of solid supports. A biotin residue is first added to the 5' terminus of one oligonucleotide and then the primer
Polymerasechain reaction techniquesGibbs 73 incorporated by PCR into the DNA fragment to be analyzed. The biotinylated strand can be conveniently captured using an avidin-coated magnetic bead, so that it may be purified from the complementary DNA prior to the sequencing [23--,39]. Solid-support sequencing has also been demonstrated using an avidin-bound column, but the beads offer greater convenience and the possibility for automation [41]. In our studies, the ability to thoroughly wash DNA bound to solid supports in order to remove salt and unwanted PCR products has improved the quality and reliability of subsequent fluorescent-automated DNA sequences.
PCR from microorganisms It is very likely that as the necessary DNA sequence information becomes ~ailable PCR will continue to displace more tedious assays for the presence of microorganisms. There have been innumerable examples of PCR-based microbe detection, but few of these represent major advances in technology. One exception is the antigen-capture polymerase chain reaction method, which has elegantly combined the specificity of the antibody-recognition of type A hepatitis virus with the sensitivity of PCR [42]. Antibodies are attached to a microtitre plate well which is then exposed to samples containing viral particles. The captured virus is retained during a washing step and then the nucleic acid of the viral genome is amplified by specific oligonucleotide primers. Finally, the PCR product is detected by gel electrophoresis. The combination of two independent methods for specific target selection will no doubt be useful for the reliable detection of many other microorganisms. The presence of conserved rRNA flanking variable sequences in most bacteria is allowing the identification of species-specific oligonucleotide priming sites [43]. First, rRNA-specific primers are used to amplify segments of DNA that contain the variable regions. The variable DNA is then sequenced and new oligonucleotide primers are generated which can be used to specifically amplify fragments from only that species. Alternatively, conserved primers can be used routinely for amplification, and another method employed to distinguish the variable segments.
PCR of synthetic oligonucleotides The use of PCR to improve the synthesis of long oligonucleotides has been elegantly demonstrated by Barnett and Erfle [44.]. The crude products of synthesis of a 234 base sequence were amplified by short PCR primers complementary to either end of the molecule. The successful amplification was monitored by agarose gel electrophoresis and the products cloned and sequenced to verify that no errors had been introduced by either chemical or enzymatic steps in the procedure. When compared with more conventional approaches of chemically synthesiz-
ing both strands of the new sequence, or of constructing partially overlapping oligonucleotides that are then copied once enzymatically, the new method is cheaper, faster and more reliable. The approach is an extremely useful addition to previous methods of 'gene synthesis' and will no doubt encourage many individuals to attempt DNA constructions that would otherwise have been too costly or time-consuming.
Contamination Finally, Sarker and Sommer [45 .°] have established the first method that does not require the usual extensive precautions against contamination of PCR by exogenous DNA fragments. Modest doses of short wavelength ultraviolet light prevent contaminating double-stranded flagments from functioning as PCR templates, but do not interfere with the ability of single-stranded, short oligonucleotides to prime the desired fragments efficiently. It can be anticipated that ultraviolet 'sterilization' of the environment where PCR mixes are constructed will become a standard procedure in laboratories where contamination is a problem.
Conclusion Innovative PCR-based methods have been developed throughout the previous year, thus expanding the list of applicaions that have been described since 1985. This trend will probably continue as the procedure is constantly adapted for use in situations where analysis or manipulation of DNA is limited by the amount or relative abundance of desired DNA fragments.
References and recommended reading Papers of specialinterest,publishedwithin the annualperiod of review, have been highlightedas: • of interest •. of outstanding interest 1.
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2.
ERLICHHA (ed): PCR Technology: Princ~oles and applicatz'ons for DNA ampUflcatio~ New York: Stockton Press, 1990.
3.
MuuasKB: The Unusual Origins of the Polymerase Chain Reaction. Sci Am 1990, 263:56-65.
4.
INNISMA, GELFANDDH, SNINSKYJJ, WHITETJ (eds): PCR Protocols. A guide to methods and a/~l#_.att~ns San Diego: Academic Press, Inc. 1990.
5.
ERLICH HA, GIBBS RA, KAZAZIANJNRI-IH(eds): The Po/ymerase Chain Reaction New York: Cold Spring Harbor Press, 1989.
6.
WHrrETJ, ARNHEtMN, ERLICHHA: The Polymerase Chain Reaction. Trends Genet 1989, 5:185--188.
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Analytical
biotechnology
7.
Gram RA: DNA Amplification by the Polymerase Chain Reaction. A n a / t ~ e m 1990, 62:1202-1214.
8.
MULLISIG Targeted Amplification of DNA by the Polymerase Chain Reaction. Annals Cloem Clinique 1990, 20.
9.
RUANOG, FENTON W, KIDD KK: Biphasic Amplification of Very Dilute DNA Samples via 'Booster' PCR. Nucleic Acids Res 1989, 17:5407.
10.
ECKERTKA, KUNKEL TA: High Fidelity DNA Synthesis by
•.
the T h e r m u s Aquatloats DNA Polymerase. Nucleic Acids Res
1990, 18:3739-3744. A thorough study of factors which are relevant to PCR conditions and which influence the ram of mutations induced by Taq DNA polymerase. 11. tto
NELSONn l , LEDBETI'ERS~ CORBO I~ VICTORIAM, RAMARIZ-SOLIS R, WEBSTERTD, LEDBZITERDH, CASKEYCT: AIu Polymerase Chain Reaction: A Method for Rapid Isolation of Human Specific Sequences from Complex DNA Sources. Proc Natl Acad Sci USA 1989, 85:6686-6690. The first description of the use of Alu-specific PCR primers for isolation of human DNA sequences. Alu PCR has since become an important tool in human molecular genetics. 12. •
ECONOMOUEP, BERGENA, WARRENAC, ANTONARAKISSE: The Polydeoxyadenylate Tract of Alu Repetitive Elements is Polymorphic in the Human Genome. Proc Natl Acad SCi USA 1989, 85:6686-6690. This paper defines the conditions for reliable amplification of fragments defined by Alu-specific primers and oligonudeotides that bind to unique sites. It also reports valuable biological observations.
Magnetic Beads as a Solid Support. Nucleic Acids Res 1989, 17:4937-4946. Magnetic beads are used as solid supports for PCR fragments by the introduction of biotin residues at the 5' end of oligonudeotide primers. The ease of manipulation of the bound fragments makes this procedure very useful for many applications. 24. •s
KEMPDJ, SM1TH DB, FOOTE SJ, SAMARASN, PETERSON MG: Colorimetric Detection of Specific DNA Segments Amplified by Polymerase Chain Reactions. Proc Natl Aca~ Sci USA 1989, 86:2423-2427. The combination of a double-stranded DNA binding-protein and PCR are used here to specifically capture and detect amplified DNA fragments. This general approach is applicable to microtitre plate formats and, therefore, may be favored for large scale screening. 25.
Izw AM, KEMP DJ: Isolating DNA Segments from Cloned Libraries Without Screening by Afl~inity Selection of PCR Products. Nucleic Acids Res 1989, 17:5859-5860.
26. •.
ROUX KH, DI-IANARMANP: A Strategy for Single Site PCR Amplification of dsDN& Priming Digested Cloned or Genomic DNA from an Anchor-Modified Restriction Site and a Short Internal Sequence. B/otechm~_~ 1990, 8:48-57. Avery clever method for performing anchor PCK This is the first report that makes use of prior internal priming in a fragment as a prerequisite for end priming, so as to give a highly specific anchor PCR strategy. 27.
RILEYJ, BUTLERR, OGILVlE D, FINNIEARR, JENNER D, POWELL S, ANAND R, SMITHJC, MARKHAMAF: A Novel, Rapid Method for the Isolation of Terminal Sequences from Yeast Artificial Chromosome (YAC) Clones. Nucleic Acids Res 1990, 18:2887-2890.
EPSTE~N, NAHOR O, SILVERJ: The 3' Ends of Alu Repeats are Highly Polymorphic. Nuc/e/c Acids Res 1990, 18:4634.
28.
ORITAM, SUZU~ Y, SEKIYA T, HAYASHI K: Rapid and SCnsitive Detection of Point Mutations and DNA Polymorphisms Using the Polymerase Chain Reaction. Genom#.s 1989, 5:874-879. The single-strand conformation polymorphism detection assay is technically the most simple method for scanning for mutations that has yet been described.
TIMBLIN C, BATIXY J, KUEHL WM: Application for PCR Technology to Subtractive cDNA Cloning: Identification of Genes Expressed Specifically in Murine Plasmacytoma Cells. Nude/c Ac/ds Res 1990, 18:1587-1593.
29. •.
13. 14. e.
15.
~(rEBERJI~ Informativeness of Human (dCMA)n(dG-dT)n Polymorphisms. Genom~s 1990, 7:524-530.
16.
AU S, WAL~CE RB: Intrinsic Polymorphism of Variable Number of Tandem Repeat Loci in the Human Genome. Nuc/e~ Act~ Res 1988, 16:8487-43496.
17.
MEULLERPR, WOLDE B: I n v i v o Footprinting of a Muscle Specific Enhancer by Ltgation Mediated PCR. Science 1989, 246:78O-786.
18.
PFEIFERGP, STEIGERWALDSD, MUELLERPR, WOLD B, R1GGSAD: Genomic Sequencing and Methylation Analysis by Ligation Mediated PCR. Science 1989, 246:780-786.
19.
ROSENTHALA, JONES DS: Genomic Walking and Sequencing by Olig~Cassette Mediated Polymerase Chain Reaction. Nucleic Acids Res 1990, 18:3095-3096.
20.
FORS ~ SAAVEDRARA, HOOD 1.. Cloning of the Shark PO Promoter Using a Genomic Walking Technique Based on the Polymerase Chain Reaction. Nucleic Acids Res 1990, 18:2793-2799.
21.
LOH EY, ELLIOTJF, CWERLAS, LANIERLL, DAVISMM: Polymerase Chain Reaction with Single Sided Specificity: Analysis of TCell Receptor Delta Chain. Sc/ence 1989, 243:217-220.
22.
FROHMANMA, DUSH MK, MARTINGR: Rapid Production of Full Lensth cDNAs from Rare Transcripts Using Single Gene Specific Oiigonucleotide Primer. Proc Nail Acad Sci USA 1988, 85:8998--9002.
23. ..
HULTMANT, STAHLL S, HORNES E, UHI£N M: Direct Solid Phase Sequencing of Genomic and Plasmid DNA Using
IX/DECKEHJ, SENGERG, CLAUSSENU, HOSSTHEMKEB: Cloning Defined Regions of the Human Genome by Microdissection of Banded Chromosomes and Enzymatic Amplification. Nature 1989, 338:348-350. Although microdissection of chromosomes is technically very difficult, PCR makes possible the efficient recovery of the minute amounts of material generated. 30. •.
KINZER~ , VOGELSTEINB: Whole Genome PCPc Application to the Identification of Sequences Bound by Gene Regulatory Proteins. Nucleic Acids Res 1989, 17:3645-3653. PCR can be used to help overcome the difficulties previously associated with screening specific DNA fragments for protein binding. 31. ..
TUERKC, GOLD I~ Systematic Evolution of Ltgands by Exponenttal Enrichment: RNA l.iwands to Bacteriophage T4 DNA Polymerase. Science 1990, 249:505-510. A report of a new approach to the study of DNA ligand interactions. 32.
HUSEX,VD, SASTRY lo IVERSON SA, KANG AS, ALTING-MEES M, BURTON DR, BENKOVICSJ, LERNERRA: Generation of a Large Combinational Library of the Immunoglobulin Repertoire in Phage Lambda. Science 1989, 246:1275-1281.
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CHAUDHARYVK, BATRA JK, GALLD MG, WIIL~GHAM MC, FrlXGERALDDJ, PASTANI: A Rapid Method of Cloning Functional Variable-Region Antibody Genes in Escherichia Coil as Single-Chain lmmunotoxins. Proc N a a Acad Sci USA 1990, 87:1066-1070.
34.
GIIJJIANDG, PERR1N S, BLANCHARDK, BUNN HF: Analysis of Cytokine mRNA and DNA: Detection and Quantitation by Competitive Polymerase Chain Reaction. Proc N a a Acad SCi USA 1990, 87:2725-2729.
35.
WANGAM, DOYLE MV, MASK DF: Quantitation of mRNA by the Polymerase Chain Reaction. Proc N a a Acad SCi USA 1989, 86:9717-9721.
36.
LIPSONKE, BASERGAR: Transcriptional Activity of the Human Thymidine Kinase Gene Determined by a Method using the
Polymerasechain Polymerase Chain Reaction and an Intron-Specific Probe. Proc Natl Acad Sci USA 1989, 86:9774-9777.
Polymerase Chain Reaction Method. Proc Natl Acad S¢i USA 1990, 87:2867-2871.
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CHEHABFF, KAN YW: Detection of Specific DNA Sequences by Fluorescence Amplification: A Color Complementation Assay. Proc N a a Acad Sci USA 1989, 86:9178-9182.
43.
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GraBSRA, NGUYENPN, CASKEYCT: Direct DNA Sequencing of Complementary DNA Amplified by the Polymerase Chain Reaction. In Methods in Molecular BiologF ec~ted by Mathew C. London: Humana press, 1991, in press.
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39.
GIBBSRA, NGUYEN PN, EDWARDS A, CIVITELLO AB, CASKEY CT: Multiplex DNA Deletion Detection and Exon Sequencing of the Hypoxanthine P h o a p h o ~ t r a n s f e r a s e Gene in Lesch-Nyhan Families. Genom/cs 1990, 7:235-244.
40.
GYLLENSTENUB, ERLICH HA: Generation of Single-Stranded DNA by the PolymeraseChain Reaction and its Application to Direct Sequencing of the HLA-DQA Locus. Proc Natl Acad Sci USA 1988, 85:7652-7656.
41.
MITCHELLLG, MERmL CR: Affinity Generation of SingleStranded DNA for Dideoxy Sequencing Following the Polymerase Chain Reaction. Anal B~cJ0em 1989, 178:239-242.
42.
JANSENRW, SIEGL G, LEMONSM: Molecular Epidemiology of Human Hepatitis A Virus Defined by an Antigen Capture
reaction techniques Gibbs
BOTrGER EC: Rapid Determination of Bacterial Ribosomal RNA Sequences by Direct Sequencing of Enzymatically Amplified DNA. FEMS Microbiol Lett 1989, 53:171-176.
BARNETtRW, ERFLE H: Rapid Generation of DNA Fragments by PCR Amplification of Crude, Synthetic Oligonucleotides. Nucleic Acids Res 1990, 18:3094. Efficient recovery of synthetic DNA longer than 50 bases is generally very diflqcult but here, PCR is used to generate the desitx~d fragment from the products of synthesis of a 234 base sequence. This process will considerably reduce the cost of 'gene synthesis' projects. 45.
SARKERG, SOMMERSS: Shedding Light on PCR Contaminatiotx Nature 1990, 343:27. ~ e simplest way yet described for getting rid of unwanted PCR contamination.
RA Gibbs, Institute for Molecular Genetics, Baytor College of Medicine, Texas Medical Center, Houston, Texas 77030, US&
75
