Polymerase chain reaction strategy.

Further

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Annu. Rev. Biochem. 1992. 61:131-56 Copyright © 1992 by Annual Reviews Inc. All rights reserved

Annu. Rev. Biochem. 1992.61:131-156. Downloaded from www.annualreviews.org by University of Alabama - Birmingham on 01/07/13. For personal use only.

POLYMERASE CHAIN REACTION STRATEGY Norman Arnheim Molecular Biology Section, University of Southern California, Los Angeles, California 90089- 1 340

Henry Erlich Human Genetics Department, Roche Molecular Systems, Emeryville, California 94608

1400

53rd Street,

CONTENTS INTRODUCTION ..... . . . . . . . .. ... . . . . . .... . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . .... . . . . .. ..... . .. . .

1 32

THE BASIC METHOD . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .... . . . . . . . . . . . .... . . . . . . . . . ..... . . . . . . . . . . . . . . . .

1 32

BIOCHEMISTRY OF THE REACTION........................................................ .

1 35 1 35 136

PCR Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Details of the Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PROPERTIES OF THERMOSTABLE POLYMERASES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

137

PRACTICAL CONSIDERATIONS................................................................

1 39 139 140 141 141

. . ��;��: !�� ::::::::::::::::::::: :::::::::::::: ::::::::::::::::::::::::::::::::: :

Misincorporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hybrid PCR Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l i i . . . : :

GENE AND eDNA AMPLIFICATION ..........................................................

Cloning PCR Product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Single-Sided DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Whole Genome PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . cDNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enriching Gene or cDNA Libraries using PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amplification with Degenerate Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

142 142 142 143 143 145 146

ALLELE-SPECIFIC PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . ... . . . . . . . . . . . . . . . . . . . . .

.

146

SEQUENCING PCR PRODUCT ..... . . . . . . . .... . . . . . . . .... . . . . . . . ...... . . . ... ....... . . . . . . . . . . ...

148

GENOMIC FOOTPRINTING ...... .......... . . . . . .... . . . . . . . . . . . ... . . . . . .. . . . . . . . . . . .. . . . . . . . . .... .

148

GENOMIC SEQUENCING ........................................................................ .

149

.

PCR SENSITIVITY .................................................................................

147

131 0066-4 154(92(0701-0 13 1$02.00

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MUTATION DETECTION AND ANALySIS..................................................

Gel Electrophoretic Methods................................................................... Methods Involving Chemical Cleavage ... ................................................... DNA AND RNA SELECTION TECHNIQUES .............. . . . . . . . . . . .. ........... ......... Nucleic A cid-Protein Interactions ................................................... .......... Nucleic Acid-Ligand Interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. ... ...... ..... .

. .

. . .

CONCLUSION

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

150 150 151 151 151 152 153


INTRODUCTION About 20 years ago, recombinant DNA technology was introduced as a tool for the biological sciences. Molecular cloning has allowed the study of the structure of individual genes of living organisms. This method depends on the replication of the DNA of plasmids or other vectors during cell division of microorganisms. In 1984, a team of scientists at Cetus Corporation developed a DNA amplification procedure based on an in vitro rather than an in vivo process. Known as the polymerase chain reaction (PCR; 1-3), this method can produce large amounts of a specific DNA fragment from a complex DNA template in a simple enzymatic reaction. Cell-free gene amplification by PCR can simplify many of the standard procedures for cloning, analyzing, and modifying nucleic acids. PCR is characterized by the three Ss: selectivity, sensitivity, and speed. Virtually pure DNA fragments from complex genomes can be obtained in a matter of hours rather than the weeks or months traditional cloning requires. The method utilizes a DNA polymerase and two oligonucleotide primers to synthesize a specific DNA fragment from a single stranded template sequence. The amount of starting material needed for PCR can be as little as a single molecule rather than the usual millions of molecules required for standard cloning and molecular biological analysis . Although purified DNA is used in many applications, it is not required for PCR, and crude cell lysates also provide excellent templates. The DNA need not even be intact, in contrast to the requirements of other standard molecular biologi cal procedures, as long as some molecules exist that contain sequences complementary to both primers (see below) . The speed and sensitivity of PCR have been widely recognized by scientists in both medicine and basic biology, and the method has been applied to problems that a few years ago were thought to be inaccessible to molecular analysis.

THE BASIC METHOD The principle of the method is shown in Figure 1. To amplify a specific DNA segment by PCR it is not necessary to know the nucleotide sequence of the

PCR STRATEGY

133

target DNA. Ideally two small stretches of known unique sequence that flank the target are used to design two oligonucleotide primers. The length of the primers (usually

2:20 bases) must be sufficient to overcome the statistical

likelihood that their sequence would occur randomly in the overwhelmingly large number of nontarget DNA sequences in the sample.

PCR is carried out

in a series of cycles. Each cycle begins with a denaturation step to render the target nucleic acid single-stranded. This is followed by an annealing step


during which the primers anneal to their complementary sequences so that their

3' hydroxyl ends face the target. Finally each primer is extended through

the target region by the action of DNA polymerase. These three-step cycles are repeated over and over until a sufficient amount of product is produced. A critical requirement is that the extension products of each primer extend far enough through the target region to include the sequences of the other flanking primer. In this way, each extension product made in one cycle can serve as a template for extension in the next cycle. This results in an exponential increase in

PCR product as a function of cycle number. After the

first few cycles the major product is a DNA fragment that is exactly equal in length to the sum of the lengths of the two primers and the intervening target DNA. The earliest peR experiments ( l, 2) utilized the Klenow fragment of Escherichia coli DNA polymerase I at a temperature of 37°C and often produced incompletely pure target product as judged by gel electrophoresis. The isolation of a heat-resistant DNA polymerase from

Thermus aquaticus

(Taq) allows primer annealing and extension to be carried out at an elevated temperature

(3), thereby reducing mismatched annealing to nontarget se

quences. This added selectivity results in the production of large amounts of virtually pure target DNA. Beginning with

1 p,g of human DNA, which 25 cycles of peR can

contains about 300,000 copies of each unique sequence,

generate up to several p,g (a few picomoles) of a specific product several hundred base pairs in length. Another important advantage of Taq polymerase is that it escapes inactiva tion during each cycle, unlike the Klenow enzyme, which had to be added after every denaturation step. This has allowed automation of

peR using

machines that have controlled heating and cooling capability. A number of thermocyclers are commercially available at relatively low cost. This de velopment has been a significant factor in the rapid application of this

1985 , more 5000 scientific papers have been published using peR (G. Mcgregor,

technology by the scientific community. Since the first report in than

personal communication). The large number of publications of course makes it impossible for us to review all of the important contributions to the development and application of

peR technology.

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ARNHEIM & ERLICH

F] t::j

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5'

----3· P1 -*

* fZ22222221 P2


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3' - GGACGTATCCTGTCCGATTGCCA- 5'

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------ 5'

Fundamental principle of PCR.

(a) DNA double helix with boxed target segment. (b)

Hybridization of PCR primers to opposite strands of the region flanking the target. The 3' end of each primer is indicated by an *. The base-base interactions of one primer (boxed) with the region flanking the target is shown below.

(e) Result of extending each primer with a DNA polymerase.

The dotted region of each extension product indicates the portion that is complementary to the other primer. (d)

peR products alter the material in (e) was subjected to another round of

amplification. Reprinted with permission of the American Institute of B iological Sciences (139).

peR STRATEGY

135

BIOCHEMISTRY OF THE REACTION


peR

Specificity

In a typical mammalian genome (with a complexity on the order of 109 nuc1eotides), a lOOO -base-pair target would represent only one millionth of the DNA. The annealing of the primers to other sequences besides the target in such complex templates could result in extension and eventually amplifica tion of nontarget sequences. Specificity is achieved by designing primers flanking the target that are of sufficient length so that their sequence is virtually unique in the genome. The specificity of the interaction of the primer with the desired template versus nontarget DNA is temperature and salt concentration dependent, and appro priate conditions must be determined empirically. The conditions of the reaction must also be compatible with full activity of the polymerase. The Taq polymerase has measurable activity at room temperature and at almost all of the temperatures up to the DNA denaturation temperature (4) . The activity at low temperatures allows extension to take place o n primer template complexes that are not perfectly matched along their whole length. For optimal specificity, the highest annealing temperature possible should be used to reduce nonspecific primer extension. Nonspecific extensions them selves are not necessarily a serious problem. However, once an extension product is present in the reaction, there is a finite chance that it could serve as a template for nonspecific extension by another primer. This would create a product that has primer sequences at each end and therefore could be ampli fied along with the desired target; if the nonspecific product is smaller than the target, it may be amplified more efficiently . It is the usual practice to set up the reaction at room temperature and to begin it with a 92-96 °C denaturation step. It has been suggested that even while the samples are being prepared primer extension by the Taq DNA polymerase could occur. At room temperature there would be little specificity to primer-template interactions. Experiments have shown that some of the nonspecific amplification products can be eliminated under so-called "hot start" conditions (5, 6). This approach keeps the sample at a temperature greater than the calculated annealing temperature for the specific primer before the reaction is started . Limiting amplification to the desired target can also be enhanced using a nesting strategy that involves two different rounds of peR. After amplifica tion with one set of primers, a small aliquot is taken and amplified in a second round with either two new primers that are internal to those used in the first round (nesting; 2) or one new internal primer and one of the original primers (heminesting; 7). This strategy works because, unlike the desired target, any nontarget sequences that were amplified in the first round cannot be further

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ARNHEIM & ERLICH

amplified with the internal target-specific primer(s) used in the second round. Recently a new approach to the heminesting strategy has been developed in which it is not necessary to carry out two distinct rounds in separate tubes. Following the first round in which all three primers are present, the thermal cycing conditions are changed so as to preferentially enhance the annealing of the nested primer (8).


Details of the Reaction PCR is quite complex even though there are a limited number of reagents used. In addition to a genomic DNA sample usually containing less than 1 amol of specific target sequence, the 25-100 JLliter volume includes 20 nmol of each of the four deoxynucleoside triphosphates (dATP, dCTP, dGTP, and TTP), 10 to 100 pmol of each primer, the appropriate salts and buffers, and DNA polymerase. The nucleotide concentration must be sufficient to saturate the enzyme, but not so low or unbalanced as to promote misincorporation (see below). The primer concentration must be high enough to anneal rapidly to the single-stranded target and, in later stages of the reaction, faster than target-target reassociation. Temperature control and timing are also impor tant. Denaturation must be efficient, but the temperature must not be too high or held for too long a period, because the Taq polymerase, although heat resistant, is not indefinitely stable (4). The temperature used for annealing must maximize specific primer annealing and polymerase elongation but not sacrifice yield by reducing primer-template hybridization. One of the advantages of PCR is its speed. The Taq polymerase can extend sequences up to 1000 bases in length in less than a minute (4). With short PCR cycle times, rapid temperature equilibration of the sample is necessary. The reaction mixture is usually overlaid with mineral oil to prevent evapora tion, thereby contributing to rapid thermal equilibration and eliminating a concentration of reagents during the course of the reaction. A newly designed thermocycler is capable of very rapid temperature change, and because the whole sample tube including the cap is heated, mineral oil is not required to prevent evaporation (9). In general, using 20-nucleotide-Iength primer se quences with a 50% GC content, denaturation at 92-96 °c for 30--60 seconds, annealing at 55-60 DC for 30 seconds, and extension at 72 °c for 1 minute is satisfactory for targets less than 500 bp. It is often found that a simple two-step cycle (95 °c denaturation; 60°C annealing and extension) also gives excellent results. If every template molecule in the sample is completely extended at each cycle then the amplification efficiency is 100%. In practice, efficiencies can vary quite significantly throughout the course of amplification. For example, the amount of product produced at each cycle eventually levels off. This plateau can be explained by two phenomena. As the concentration of double-


PCR STRATEGY

137

stranded product reaches high levels, competition increases between anneal ing of template (PCR product) to primer and reannealing of the com plementary template strands. Secondly, the amount of enzyme is finite and eventually there is not enough to extend all of the primer-template complexes in the allotted time. The efficiency may also depend upon the amount of original target and on properties of the target that are not well understood such as the likelihood of secondary structure in the single-stranded template. In general, long targets are less efficiently amplified than short ones, but there are exceptions to this generalization, which could be due to the actual sequences involved. Finally, some primers seem to work better than others even when the target sequence is the same, but the rules that govern this phenomenon are not yet known. The addition of reagents that tend to reduce secondary structure formation can in some circumstances improve amplifica tion ( 1 0, 11). The average efficiency of a series of PCR cycles can be described by the following equation: N = n (1 + Er, where N the final amount of product, n = the initial amount of target, E the efficiency of amplification, and c = the number of PCR cycles. Because of the plateau effect, the measurement of the efficiency of peR in its exponential phase must be carried out when only small amounts of product are present. The reported average efficiencies are usually greater than 0.60 and can be as high as 0.95 (3, 12-14). In general, peR has been used as a tool rather than being the object of detailed investigations of its complex reaction dynamics. Trying to un derstand the nature of the structure of the nucleic acids, the protein-nucleic acid interactions, and the fundamental enzymology and structure of the thermostable polymerases could lead to important advances in this tech nology. =

=

PROPERTIES OF THERMOSTABLE POLYMERASES As discussed above, the introduction of a thermostable DNA polymerase from Thermus aquaticus (Taq polymerase) into the PCR greatly simplified the PCR protocol and allowed the development of simple thermal cycling instruments to automate the reaction. It also dramatically increased the specificity and yield of the PCR by allowing primer annealing and extension to be carried out at higher temperatures. The Taq DNA polymerase isolated from the cloned Thermus aquaticus gene expressed in E. coli (15) has a specific activity of 200,000 units/mg and an inferred molecular weight of 93,910 (4). It has a temperature optimum of 75-80 DC, depending on the DNA template. Under appropriate conditions, it is highly processive and has been reported to have an extension rate of >60 nucleotides per second at 70°C using M13 phage


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ARNHEIM & ERLICH

DNA as template (16). At lower temperatures, the extension rate and the extent of processivity are both reduced. Taq polymerase lacks a 3'-5' exonuclease activity or "proofreading" function but does contain a 5'-3' exonuclease activity during polymerization (16-19). A primer annealed to a single-stranded template will be degraded from its 5' end by polymerase extension of a second primer annealed to the template 5' of the first primer. This activity has recently been developed into an assay for detecting the presence of specific DNA templates in a sample (20). G enetically engineered variants of the Taq enzyme lack this activity but carry out polymerization (8). Under some conditions, the Taq DNA polymerase is capable of adding a single nontemplate-directed nucleotide (preferentially dATP) to a blunt-ended duplex DNA fragment (21). This activity, common to other polymerases that lack the 3'-5' proofreading exonuclease, can pose problems for blunt-ended cloning of PCR products. This activity may also be involved in the formation of the so-called "primer-dimer," a double-stranded PCR artifact consisting of the two primers and their complementary sequences (22) and, occasionally, additional bases inserted between the primers. The formation of "primer dimer" probably occurs when one primer is extended using the other primer as a template, and would be expected to be dependent on primer sequence, primer concentration, and enzyme concentration. Primer-dimer usually is seen in PCR experiments involving high cycle numbers, such as those ne cessitated in amplifying rare targets. When primers are inadvertently designed with partially complementary 3' -termini, primer-dimer formation appears to be more frequent. The amount of primer-dimer formed is inversely pro portional to the amount of the target PCR product because of enzyme compe tition between the various DNA molecules being replicated. Efficient primer dimer amplification reduces the sensitivity of specific target analysis. The initial formation of these products can occur at ambient temperatures; "hot start" (see above) eliminates this phase of the thermal profile and, con sequently, can reduce the accumulation of "primer-dimer. " Recently, a variety of thermostable DNA polymerases with different prop erties have been isolated from other bacteria. One, from the thermoacidophil ic archebacterium Sulfolobus acidocaldarius, has been shown to carry out polymerization at 100°C (23-25), which could facilitate the amplification of regions of high secondary structure and enhance specificity. In the case of Taq polymerase, the enzymatic incorporation of modified bases such as 7-Aza· dGTP has proved useful in the amplification of sequences with secondary structures in GC-rich regions (26). Some of the new thermostable polymerases may allow the efficient amplification of larger PCR products (E. Rose, personal communication). The introduction of thermostable accessory proteins may also prove helpful in increasing the processivity of polymerases · during PCR and allow the amplification of longer products.


peR STRATEGY

139

The search for new thermostable polymerases has resulted in the discovery of one with reverse transcriptase activity (27). A polymerase from Thermus thermophil/us can reverse transcribe RNA efficiently in the presence of MnClz. The DNA polymerase activity of the same enzyme can be stimulated by chelating the MnCl2 and adding MgCh, allowing cDNA synthesis and PCR amplification to be carried out in a single-enzyme, single-tube reaction. The thermostability of the enzyme could minimize the effect of secondary structure in the RNA template and allow efficient cDNA synthesis at high temperatures. The topic of cDNA amplification is further discussed below. Finally, polymerases from Thermoplasma acidophilum, Thermococcus litoralis, and Methanobacterium thermoautotrophicum have been reported to have 3'-5' exonuclease activities (28-30). The consequences for mis incorporation during polymerization are discussed below. PRACTICAL CONSIDERATIONS While PCR provides a very powerful tool for molecular analysis, the un initiated investigator should be aware of some aspects of the procedure that should be considered in the design and interpretation of experiments.

Misincorporation The rate of nucleotide misincorporation during the PCR has been estimated in various studies by determining the frequency of misincorporated nucleotides in the sequence of cloned PCR products and calculating an average rate per cycle (3, 31, 32). The misincorporation rate would be expected to reflect not only the properties of the polymerase itself (e.g. presence or absence of 3 '-5' exonuclease proofreading activity), but also the reaction conditions. Bio chemical fidelity measurements of "nonproofreading polymerases" (e.g. AMV reverse transcriptase and Drosophila melanogaster DNA polymerase a) revealed that the nucleotide misincorporation is dependent upon the dNTP concentration (33). The initial estimate for nucleotide misincorporation (1. 7 X 10-4 nucleotides polymerized per cycle) by Taq polymerase during PCR (3) used 1.5 mM of each dNTP, 10 mM MgCI2, and a 37°C annealing temperature to amplify a 272-base-pair fragment from the HLA-DPBl gene. More recent studies (31, 32) gave an average nucleotide misincorporation rate of 5 x 10-6 per cycle. These studies used lower dNTP and MgCh con centrations (200 JiM of each dNTP and 1.5 mM MgCI2) as well as higher annealing temperatures (54-55°C). In general, the rate of misincorporation as well as that of extension from a 3' mispaired primer may be reduced by minimizing the annealing/extension time, maximizing the annealing tempera ture, and by minimizing the dNTP and MgCl2 concentrations (34). However, reaction conditions that maximize fidelity may reduce PCR efficiency. Also, although the calculations yield an "average" misincorporation rate per cycle,


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the fidelity of thennostable polymerases may vary during the PCR (4). The misincorporation rate of thennostable polymerases with 3'-5' exonuclease proofreading activity is likely to be lower. A comparison of the fidelity of the Taq and T. litoralis polymerases shows that the latter enzyme, which has proofreading activity, may misincorporate at 25% of the rate of the Taq enzyme (30). For most PCR applications, such as direct sequencing (see below) or characterization by hybridization probes or restriction enzyme digestions, it is the population of amplified products that are analyzed and therefore, rare misincorporated nucleotides do not pose any problem in the interpretation of experiments. The actual fraction of amplified molecules that contain a muta tion is related to the number of bases in each target molecule, the rate of misincorporation per base per cycle, and the number of cycles. A mathemati cally precise theory for estimating the frequency of molecules without any misincorporations has recently been put forward (35). Using the most recent average estimate of misincorporation (8. 5 x 10-6; 31, 32), only 3% of the molecules of 200-base-pair peR product will contain a misincorporated base somewhere in the sequence after 30 cycles. Of course most of the molecules with misincorporated nucleotides would not contain the same mutation. Addi tional estimates of misincorporation are required since the rates might vary for the particular sequence studied and the actual PCR conditions used. While misincorporations appear to have relatively little effect on analysis by most methods, the detenninations of individual cloned sequences derived from peR products of course reveal such rare errors. It is advisable either to sequence multiple clones derived from a single PCR or to clone the products from several independent amplification reactions to identify any potential errors.

Hybrid peR Products When the sample being amplified is heterozygous (two different alleles for the target locus) or when many different but related sequences are amplified with the same primers (e.g. a multigene family), in vitro recombination or tem plate-strand switching is theoretically possible. Although a very rare event, PCR hybrid sequences can be fonned when a primer that has been partially extended on one of the templates is annealed and extended on a similar template in a subsequent cycle (3, 36-38). For this so-called "jumping" to occur, the concentration of partially extended PCR primers must be quite high. The occurrence of very rare hybrid PCR products. amplified from genomic DNA was initially discussed in Saiki et al (3). The: model experiments that studied this phenomenon with plasmid DNA, howev·· er, used high concentrations of template, thereby increasing the likelihood that the levels of partially extended primers would be sufficient to promote:


peR STRATEGY

141

jumping (36-38). The likelihood of partial primer extension is also dependent on the distance between the primers, secondary structure, the time allowed for polymerase extension, the processivity of the polymerase, and the extent to which the original target DNA sequences contained single strand breaks. Again, most methods of analysis characterize the population of PCR prod ucts, not rare individual hybrid products. In general, the formation of hybrid molecules can be minimized if the minimum number of PCR cycles required for product detection are carried out. Under some circumstances jumping PCR may be beneficial (36). Assume a sample of DNA is highly degraded and no completely intact target molecules exist. If the DNA contained a set of partially overlapping fragments from the target, then repeated jumping could eventually produce a full-length PCR product.

Preferential Ampl ification Under some circumstances preferential amplification of one allele relative to the other can occur in heterozygous samples. One possible mechanism for differential priming is that one of the two alleles may have a rare DNA sequence polymorphism in the primer-binding site. Another potential mech anism is that one allele could have a higher GC content, and therefore require a higher denaturation temperature to generate functional PCR template than does the other allele. In the case of length polymorphisms detected as PCR products of different size, the smaller allele can, under some circumstances, be amplified preferentially. In addition, one allele in a heterozygous sample can be amplified preferentially due to stochastic fluctuation when sampling a very low number of target molecules.

Contamination Contamination of sample tubes by PCR product derived from earlier ex periments can cause serious problems. Amplified DNA from a single sample can contain several picomoles of PCR product. Even one millionth of that product could have disastrous consequences if it contaminated a I-microgram sample of genomic DNA containing 300,000 copies of a unique sequence. When carrying out PCR experiments, aerosol formation and other possibili ties of contamination must be minimized. Physically separating the areas in the laboratory where PCR reactions are set up from those where the PCR products are analyzed can be of significant help. A set of pipettes that are dedicated to setting up PCR reactions is also warranted. Reagent solutions should be aliquoted. Micropipette tips with built-in filters can also be useful. A number of other precautions in the technical aspects of carrying out PCR are discussed in Kwok & H iguchi (39). Recently, several protocols for eliminating contaminating DNA sequences


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ARNHEIM & ERLICH

carried over from previous reactions have been developed. UV irradiation of the reaction tube contents can damage any contaminating sequences before the DNA template and polymerase are added (40). Another approach uses dUTP instead of dTTP for PCR (41; J. J. Sninsky et ai, in preparation). PCR products therefore are susceptible to degradation by the enzyme uracil N glycosylase. Before beginning a peR experiment, a sample is treated with the glycosylase, which will destroy any carried-over PCR product but will not degrade the desired genomic DNA template. Following heat inactivation of the glycosylase, PCR is carried out with dUTP. In a still different approach to the contamination problem, a photochemical reagent is added before the reaction and activated after peR is completed. The reagent will cross-link the two strands of the PCR product and render them unamplifiable (42). GENE AND eDNA A MPLIFICATION

Cloning peR

Product

PCR produces large amounts of product derived from most if not all of the copies of the target present in the original sample, but individual product molecules can be cloned by recombinant techniques. Cloning can be sim·· plified if the primers used for PCR are constructed so that sequences contain ing a restriction endonuclease cleavage site are appended to their 5' end (43). Following restriction enzyme digestion, the peR product has "sticky ends," which can be ligated to an appropriately digested cloning vector. peR product can also be cloned following blunt-end ligation, although nontemplate·· directed nucleotide addition (see above) can lead to lower efficiency. Recently, a strategy for introducing "sticky ends" into the product and vector by appropriately designed peR primers has eliminated the need for restriction enzyme digestion and in vitro ligation (44).

Single-Sided DNA Amplification INVERSE peR The basic method of peR requires that DNA sequence information be available on both sides of the target sequence. Inverse PCR allows amplification of targets where precise DNA sequence information is available on only one side (45-47). If two primers are designed in the known region so that, unlike conventional PCR, they face away from each other, the flanking sequences can be amplified if a DNA fragment containing the known and flanking sequences is first circularized by ligation following restriction enzyme digestion.

LIGATION-MEDIATED PCR Inverse peR can be difficult to carry out, pri marily because self-ligation of the ends of linear DNA fragments in complex genomes is rather inefficient. A different approach to single-sided amplifica-

PCR STRATEGY

143

tion involves the addition of a known sequence, in the form of a linker, to one end of a duplex DNA fragment whose other end has sufficient sequence information available to allow a primer to be made (48-50). The linker is added by DNA ligation under normal conditions. One elegantly designed primer for this purpose (48),


5'

OH-GCGGTGACCCGGGAGATCTGAATTC -OH OH - CTAGACTTAAG-OH 3'

3' 5' ,

has the following important attributes. One end is a blunt-ended duplex, while the other is single stranded. Neither of the 5' ends is phosphorylated. Thus, the linkers cannot ligate to each other and only the long strand will be ligated to the blunt end of a genomic DNA fragment. The short strand is AT rich compared to the long strand and thus, after ligation, an appropriate tempera ture can be found that will prevent the two linker strands from annealing. PCR can be carried out using the long linker sequence as one primer and a genomic sequence from the other end as the other primer. Sequences designed to be used as primer sites can also be added to the 3' end of a target sequence by enzymatic extension using terminal transferase (see below).

Whole Genome peR Another application of the linker ligation strategy allows amplification of every DNA sequence present in a complex mixture . While this approach lacks specificity, it is useful when starting out with a very small amount of a complex DNA and large amounts are desired for some purpose. For example, DNA libraries have been constructed starting from specific segments of c hro mosomes dissected from whole chromosomes on a microscope slide (51, 52). After dissection, the DNA is digested with a restriction enzyme and the fragments ligated to a plasmid. Plasmid sequences flanking the insertion site are used for PCR primers (51). Whole-genome PCR can also be applied to the analysis of protein-DNA interactions (see below).

cDNA Amplification Amplification of cDNAs where sequence informa tion permits primer design at both flanking regions is straightforward. Total cDNA can be made with a reverse transcriptase (R T) using standard pro cedures including oligo dT or random hexanucleotide primers (53). Amplification of a specific cDNA with the appropriate message specific primers is then carried out (54). Whenever possible, primers used for amplify ing cDNAs of RNAs should not be positioned within the same exon . This strategy will minimize amplification from small amounts of contaminating DNA (55) since, if the primers lie within the same exon, the size of the PCR GENERAL APPROACHES

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product produced from genomic DNA would be indistinguishable from the product expected from the cDNA target. If a nonprocessed RNA is the peR target, RNase-free DNase I digestion should be employed to destroy possible contaminating DNA sequences and confirmed with appropriate controls, including samples amplified without a prior reverse transcription step. The fact that primers can be designed for any pair of exons led to an early application of cDNA peR in the analysis of RNA processing (56). Several approaches using peR to quantitate the amount of specific mRNAs have been published. The final amount of peR product depends not only on the initial amount of mRNA but also on the efficiency of reverse transcription and peR. Two basic approaches have been used. In one, two different cDNAs are amplified and, from the ratio obtained, absolute levels of one can be calculated if already known fur the other, assuming that the two cDNA targets are amplified at similar efficiencies (14). An alternative approach (57) involves spiking each sample with a known amount of a control DNA sequence. The control sequence is designed to be as similar in sequence as possible to the cDNA target so that they will have identical amplification efficiencies using the same pair of primers. The target and control can differ by as little as a single nucleotide substitution that alters a restriction enzyme site. Since each unknown sample receives a known amount of the control sequence, the absolute amount of the target can be determined by comparing the levels of the control and target products pro duced in the same peR reaction after restriction enzyme digestion. While this method should be accurate for quantitating cDNA levels, it does not take the: efficiency of reverse transcription into consideration. However, Wang et al (58) spike the sample with an RNA transcript from a plasmid containing a modified target DNA. A known amount of the RNA transcribed from the plasmid is added to the sample as a control sequence.


mRNA QUANTITATION

SINGLE-SIDED eDNA AMPLIFICATION In some cases the available sequence information may not permit both flanking primers to be designed for cDNA amplifiation. Two examples of this are T cell receptor and immunoglobulin mRNAs where a 3' constant region suitable for primer design is associated with 5' variable regions of unknown sequence. The linker ligation strategy cannot be used because the product of reverse transcription using a primer from the 3' end of the message, although blunt-ended, is an RNA-DNA hybrid and therefore not a substrate for DNA ligase. peR of RT copies of specific RNAs where only one primer sequence is available, however, can be carried out using "single-sided" or "anchor" peR (59-61). Assume that specific sequence information for primer construction is only available at the 3' end of the messenger RNA. Following reverse transcription, the 3' end of the cDNA is modified by addition of nucleotides with terminal deoxy-

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nucleotidyl transferase. The modified cDNA product containing, for ex ample, a string of dOs at the 5' end (60) is a substrate for second strand synthesis with an "anchor" primer containing, in addition to a poly-C region at its 3' end, a unique sequence at its 5' end. PCR is carried out with a primer containing the unique sequence portion of the "anchor" and the original message-specific primer from the 3' end. If specific sequence information is known only at the 5' end of the RNA, an oligo dT containing anchor primer can be used for the reverse transcription step at the 3' poly A tail. In analogy with whole genome PCR, it may be advanta geous in some circumstances to amplify all of the cDNAs derived from an RNA preparation. For example one may want to make a cDNA library from a very small amount of starting material. In this case a primer specific for the poly A tract and one specific for whatever nucleotide was added to the 3' end of the eDNA by terminal deoxynucleotidyl transferase can be used for amplification (62-64). eDNA LIBRARIES

Enriching Gene or cDNA Libraries using peR Methods of using PCR to enrich a cDNA (65-67) or genomic DNA (68) sample for specific classes of sequences have recently been devised. Although these enrichment strategies are primarily based on standard molecular biologi cal practice, PCR enables very small amounts of starting material to be used. Conventionally produced double-stranded cDNA can be amplified after liga tion of a specific linker to both ends (65). In this case single-stranded cDNA from a mouse plasmacytoma cell line was first subjected to subtractive hybridization with RNA from a B cell lymphoma line. Following second strand synthesis and linker ligation to both ends, the selected cDNA was amplified. Low-abundance cDNAs specific to the plasmacytoma line were eventually cloned. PCR can be used as an approach to construct a "normal ized" cDNA library (66-68). The advantage of such a library is that all of the cDNAs would be present at about the same concentration, allowing cDNAs of very rare messages to be cloned with the same efficiency as highly abundant ones. In one strategy (67) cDNAs produced by conventional methods using random hexanucleotide primers are first cloned into a bacteriophage library and the inserts are amplified using PCR primers from the vector. The PCR products are then denatured, allowed to reassociate for various time periods, and the remaining single-stranded PCR product purified. Using an appropriate reassociation time decreases the amount of highly abundant cDNA sequences present in the single-stranded fraction relative to the rare cDNA sequences. Following PCR using the single-stranded sequences as template, the product is recloned into a vector. These methods could allow cloning of very rare but developmentally important cDNAs.

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Amplification with Degenerate Primers

Although perfect complementarity is usually desirable in the design of peR primers to maximize specificity, it is not absolutely required for successful peR. Primer-template annealing under less stringent conditions allows suc cessful peR, although the products may be less homogeneous. Sometimes this can provide a distinct advantage. For example, sequence information from a known gene can be used to design primers to amplify a heretofore undiscovered but related gene. If the related gene has some regions of amino acid sequence identity with the known gene, a mixture of primers based on the sequence of the known gene can be used for peR in a manner similar to the standard practice of using mixed oligonucleotide probes for screening cDNA libraries by hybridization. For peR, two primer mixtures, one for each flanking sequence, are required. Each primer mixture needs to be composed of all of the different nucleotide sequences that can code for the same amino acid sequence and therefore is expected to contain at least one primer per fectly complementary to the coding sequence of the related gene as well as a number of primers differing only by one or a few bases. Primer mixtures containing more than 200,000 different sequences have been used suc cessfully (69). This approach is especially useful in the search for members of gene families with similar physiological functions. Based upon protein se quence data, new members of a number of protein families have been discovered (61, 70-74). At the extreme, peR makes it possible to clone specific cDNAs even in the absence of any direct nucleic acid sequence information. Using only protein sequence data, degenerate primers can be successfully utilized (75). Degenerate primers have also been used as an approach to detecting uncharacterized viruses related to viruses that are already known. Mack & Sninsky (76) used a H epadnavirus model system to demonstrate the utility of this method. Using a similar procedure, sequences related to retroviral reverse transcriptases in human DNA have been detected (77-79). ALLELE-SPECIFIC PCR The basis for many of the peR modifications mentioned above is that single base-pair mismatches between primer and template do not necessarily prevent the production of the desired peR product. H owever, under some circum stances, even a single mismatch between primer and template can prevent amplification. In fact, there are a number of situations in which such specific ity is desirable. For example, alternate forms of a single gene (alleles) that differ by a single substitution can exist in a popUlation, and amplification of only one may be desired. Allele-specific peR relies on the fact that a base-pair mismatch at the 3' end of a primer not only reduces the efficiency of


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extension by the polymerase, but can also reduce the thermal stability of the primer-template complex (80--83). The extension of a primer mismatched at the 3' end by a DNA polymerase lacking a 3' to 5' exonuclease activity can be dramatically reduced by lowering the total dNTP concentration by almost a factor of 100 (7, 83). This effect of dNTP concentration is expected based on the fundamental enzymology of mismatch extension (84). The use of lower dNTP levels, however, imposes some constraints on overall amplification efficiency. Recent studies on the effects of mismatches at and near the 3' end of peR primers provide important information for the design of allele-specific primers (85).

peR SENSITIVITY The sensitivity of peR is based primarily on two factors: (a) the number of target molecules present in the sample and (b) the complexity of the nontarget sequences. The detection of specific peR product amplified from a unique sequence gene present in one or a few cells requires a great many cycles. As a result, many more opportunities exist for nontarget amplification and primer dimer formation, both of which ultimately reduce sensitivity because of competition for available peR reagents (e.g. enzyme). "Hot start" is one strategy that could minimize these artifacts; a nesting strategy is another. Amplification of a single-copy gene in a single haploid cell can produce virtually pure peR product as detected by gel electrophoresis (7). The analy sis of single sperm for genetic mapping indicates that the probability of amplifying the desired unique sequence to a detectable level ranges between 70% and 95% (86-88). Single-cell peR has also been applied to the analysis of oocytes and polar bodies in preimplantation genetic disease diagnosis (see 88), and characterization of immunoglobulin gene rearrangements in memory B cells (89). peR also makes it possible to study RNAs present in one or a few cells. peR of cDNA has already been used to search for the presence of specific messenger RNAs in the earliest stages of mouse embryogenesis (90). Messen ger RNAs of various growth factors from small numbers of macrophages isolated from wounds have also been examined in an effort to better un derstand the healing process (91). An interesting observation from studies on cDNA amplification is that some cells not expected to transcribe certain genes may have very low levels of so-called "ectopic" or "illegitimate" transcripts (92, 93). This finding can be of practical use in the analysis of mRNAs normally found in tissues that are not readily accessible. For example, mRNA for the musc1e- and brain-specific protein dystrophin, which is altered in Duchenne muscular dystrophy, can be amplified from more readily

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accessible white blood cells. Using this source of DNA from patients with the disease, many new dystrophin mutations have been discovered (94). A problem similar to single-cell analysis exists when the number of target molecules is greater than one but the ratio of target DNA sequences to total DNA is significantly less than that typical for a unique sequence gene in a mammalian genome. For example, detecting the presence of a few viral genomes in a sample containing millions of cells poses similar sensitivity problems and calls for many of the same solutions described above (95). SEQUENCING PCR PRODUCT A number of different strategies have been devised for directly sequencing PCR product (96- 100). Double-stranded DNA product can be sequenced immediately after removing unincorporated dNTPs and primers by standard methods and has the advantage that the effect of misincorporations is elimi nated. A modification of the standard PCR protocol, called "asymmetric" PCR, has been introduced to facilitate direct sequencing ( 10 1). In this pro cedure unequal amounts of the two primers are used. One primer is eventually used up, and the remaining primer continues to be extended each cycle so that an excess of one of the strands of the PCR product is generated. Additional methods have been developed to produce single-stranded template (102, 103). A lthough standard dideoxy dNTP sequencing methods work well, a chemical degradation method for directly determining the sequence of amplified DNA fragments that have incorporated phosphorothioate nucleotides during PCR is also possible ( 104). Carrying out PCR in the presence of low levels of dideoxy dNTPs has also been reported as a shortcut to DNA sequencing (105, 105a). In diploid organisms where allelic differences might exist, the PCR product of a single gene could be heterogeneous with respect to DNA sequence. Direct sequencing of double-stranded PCR product from heterozygotes has been shown to be able to identify those positions at which a polymorphism exists ( 100). For some loci, however, alleles differ by multiple substitutions. It is impossible to know, therefore, which of the alternative bases at each polymorphic position are associated with which alleles by sequencing pooled PCR product. A number of different strategies to overcome this problem have been discussed by Gyllensten (106). GENOMIC FOOTPRINTING The ability of PCR to increase the concentration of rare DNA fragments in a complex mixture has been adapted to the analysis of DNA-protein interactions in living cells (genomic footprinting). The goal of this procedure is to determine the exact contacts between DNA bases and specific proteins in the


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living cell, in contrast to DNA footprinting, which determines these in teractions in vitro. The PCR approach devised by Mueller & Wold in 1 989 (48) has a number of steps in common with the original genomic footprinting method ( 107 , 108), but is more rapid and sensitive. Cells are treated with the methylating reagent dimethyl sulfate (DMS), which reacts with guanosines that are not protected by bound protein. DNA is isolated from the cells and treated with piperidine, which induces single-strand breaks in the phospho diester bond at the position of all modified guanosines. The DNA is de natured, annealed to a specific primer downstream of the potential protein binding site, and extended upstream using a DNA polymerase. The extension products are expected to terminate at positions of piperidine cleavage. The double-stranded fragments are ligated to a linker as described in the section on LIGATION-MEDIATED PCR. After denaturation, PCR is initiated using a second downstream gene-specific primer and the linker fragment. The PCR products span the region from the gene-specific primer to the end of the linker attached

at each piperidine cleavage site. Finally, a third radioactivity labeled primer is used to extend the PCR fragments and the products are run on a sequencing gel. Compared to a naked DNA sample, the absence of a fragment in a DNA sample from DMS-treated cells indicates the position of a guanosine that was protected from methylation by bound protein. Ligation-mediated PCR has also been shown to be effective in producing in vivo footprints using DNase I in place of DMS ( 1 09). GENOMIC SEQUENCING G enomic sequencing by PCR ( 1 10) is a modification of the procedure origi nally developed by Church & G ilbert ( 1 1 1 ), and uses the chemistry of purine and pyrimidine modification and cleavage developed for DNA sequencing ( 1 1 2). The application of PCR to this procedure has greatly reduced the amount of material and time and effort required. DNA is subjected to the chemical cleavage reactions followed by denaturation, primer extension, linker-ligation, and amplification using the methods described above for genomic footprinting. In the final step, the fragments are run on a DNA sequencing gel and, following electroblotting to a nylon membrane, hybrid ized to a probe specific for the gene under investigation. Since the presence of 5-methyl cytosine in the original chromosomal DNA will result in a gap in the C ladder on the sequencing gel, the position of every 5-methyl cytosine in the sequence can be determined. The method has been successfully applied to the analysis of the in vivo methylation state of several genes ( 1 10, 1 1 3 , 1 14). Previous methods of determining the methylation state relied on Southern blotting and the use of restriction enzymes, which limited the analysis of 5-methyl cytosines to specific restriction enzyme sites.


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Finally, a highly novel application of the genomic sequencing strategy has been developed to analyze sequence-specific DNA damage in vivo ( 1 1 5) . When cells grown in culture are exposed to UV irradiation, pyrimidine (6-4) pyrimidone photoproducts are formed. DNA is isolated from treated cells and strand breakage initiated at the sites of photodamage by heating in piperidine. These lesions can be studied at specific sites in any gene using specific PCR primers as described above for the genomic sequencing and footprinting strategies. The authors also propose that the repair of this damage in living cells could be studied using the same methodology. This protocol could provide very important information concerning DNA repair mechanisms. MUTATION DETECTION AND ANALYSIS peR can play a major role in discovering the molecular basis for new mutations, since it is much simpler to analyze PCR-amplified segments from organisms carrying specific mutants rather than having to clone each gene fragment using recombinant techniques.

Gel Electrophoretic Methods Denaturing gradient gel electrophoresis (DGGE) was introduced several years ago and makes use of the fact that two double-strandcd DNA fragments that differ only by a single nucleotide substitution can have different thermal denaturation profiles (116, 117). Although this difference is subtle, it can be detected by running the two DNA samples in adjacent lanes of a polyacryla mide gel that has a gradient of a DNA denaturant along the axis of DNA migration. When the fragments reach the position' in the gel where the concentration of denaturant is sufficient to cause DNA melting, their mobility is severely retarded. If one fragment has a lower melting temperature than the other it will not migrate as far. Although DGGE was originally developed for the analysis of cloned DNA segments, peR products are easily adapted to this method ( 18). A recent innovation of the method involved attaching a GC rich stretch ("clamp") to one of the peR primers (1 19). H aving a very stable structure at one end of the peR product enhances the ability to detect all possible single-base substitutions . Two other new approaches to mutation detection by electrophoresis have been developed. One, thermal-gradient gel electrophoresis, makes use of a temperature as well as chemical gradient within the gel (120). Another method is based upon being able to detect subtle conformational differences due to a nucleotide substitution by analyzing the electrophoretic mobility of PCR products that are rendered single stranded (single strand conformational polymorphisms; SSCP) and run on nondenaturing polyacrylamide gels (121, 122).

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Methods Involving Chemical Cleavage Another approach to mutation analysis is based upon the detection of base pair mismatches in heteroduplex DNA using either chemical or enzymatic methods. PCR product from a wild-type allele, when denatured and annealed to the product of a mutant allele, will form double-stranded DNA containing a base-pair mismatch. In one method, hydroxylamine is used to chemically modify mispaired Cs or osmium tetroxide for mispaired Ts ( 1 23-125). Following piperidine cleavage, the position of the mismatch can be de termined by sizing single-stranded DNA fragments on a gel. This method has, for example, been recently applied successfully to the analysis of Factor IX mutations associated with hemophilia B ( 1 26) .

DNA AND RNA SELECTION TECHNIQUES

Nucleic Acid-Protein Interactions In the past several years, there has been an explosion in the detection of DNA sequence motifs involved in the binding of transcription factors. The finding of similar motifs in different gene segments has suggested that these proteins could be involved in regulating the expression of many genes or gene fami lies. In some cases the proteins themselves have been cloned. Once cloned, the challenge is to identify what other genes have the same binding motifs. Unfortunately, this can not be done at the level of nucleic acid hybridization, because the length of the critical binding sequences is too short to allow specificity. An alternative approach based on PCR, devised by Kinzler & Vogelstein ( 1 27) , was demonstrated in an analysis of the transcription factor TFIIIA, which is involved in the regulation of 5S RNA gene transcription. Their strategy began with the shearing of total genomic DNA and ligation of "catch linkers" to each fragment. Following exposure to purified TFIIIA protein, any DNA bound was immunoprecipitated with anti-TFIIIA antibod ies. The DNA extracted from the immunoprecipitate was amplified by PCR using primer sequences on the ligated linkers. Following additional rounds of protein binding, immunoprecipitation, and amplification, the DNA fragments were cloned and characterized. The selected sequences showed a significant enrichment of those with the TFIIIA-binding motif. This technique was subsequently used to detect human genomic sequences that bound a zinc finger protein, which is amplified in a number of different tumors ( 1 28). This approach could contribute significantly to our understanding of how specific transcription factors can coordinate the expression of unlinked genes. PCR is required for the above approach to studying DNA-protein in teractions, because the level of nonspecific binding can be quite large and the concentration of true DNA-binding sites so low. Without peR, mUltiple rounds of selection would result in extremely low yields of bound DNA


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fragments , which would be substantially contaminated. Because peR can increase the absolute amount of precipitated DNA after a round of selection, even sequences with relatively small binding constants could eventually be enriched and characterized. Similar principles have also been applied to the problem of understanding the strict nucleotide sequence requirements for the binding of a protein to a DNA motif. Traditionally, this information has come from mutagenesis experiments in which specific base substitutions in small DNA fragments have been made and analyzed by their effects on protein-DNA binding using the gel retardation and DNase I footprinting procedures ( 129-131). These experiments are laborious, and each synthetic oligonucleotide is usually studied individually. Three groups have independently devised a PeR-based method for studying this problem, which has a number of advantages ( 132134). A synthetic double-stranded oligonucleotide is made so that it contains at each end a specific sequence that can be used for amplification. Internal to these primer sequences is a stretch of n nuc1eotides, which are chosen at random so that the collection of molecules contains 4n different sequences. Protein is incubated with the oligonucleotide, subjected to electrophoresis. and the retarded band used as a substrate for peR. The gel retardation step enriches for those sequences that are capable of binding the protein and the peR step produces enough DNA so that additional rounds of selection and finally characterization can be carried out. RNA-protein interactions can also be studied using the same basic strategy ( 135). To create the pool of RNAs used for selection , a DNA template containing a randomized sequence is transcribed by a bacteriophage RNA polymerase. Following each round of selection. the RNAs are reverse tran scribed, amplified by peR , transcribed by T7 polymerase, and subjected to the next round of selection.

Nucleic Acid-Ligand Interactions The study of nucleic acid binding is not limited to the interactions with proteins. Szostak and collegues ( 136, 137) developed a method to select RNAs that specifically interact with column-bound organic dyes that mimic metabolic cofactors . They independently developed a strategy for producing the random pool of RNA sequences and amplifying those that are selected on the column. The developers of these PeR-based selection techniques visualize a number of additional important applications . Selection of RNA species that bind transition-state analogs could lead to the production of novel ribozymes ( 136). The isolation of RNAs that could inhibit a step in the life cycle of an infectious agent could have therapeutic potential ( 135). Finally, it may be possible to select for RNAs that code for peptides or proteins with desired

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functions ( 1 35) . This could theoretically be accomplished b y creating a pool of mRNA variants, precipitating those RNAs that are on ribosomes followed by selecting for those nascent polypeptides that have the function desired.


CONCLUSION The speed, specificity, and sensitivity of PCR, along with the ease with which it can be carried out and its versatility, make it ideally suited for application to many problems in biology. Many recent reviews and books indicate the extent to which PCR has had an impact on fields in addition to molecular biology, including human genetics, immunology, forensic science, evolutionary biolo gy, and ecology and population biology (8, 88, 1 3 8- 145) . In addition to allowing the application of molecular biological procedures to scientific disciplines that had not exploited these techniques, other applications of PCR have produced information that was impossible to obtain previously. Among these applications include studies on RNA and DNA sequences present in individual cells (86-89, 9 1 ) retrospective medical studies on DNA in patho logical samples stored for decades as formalin-fixed paraffin-embedded tis sues without losing the histological information contained therein ( 1 46-148), and analysis of DNA from tissues of extinct plants and animals ( 149-1 5 1) . Using PCR in the selection o f nucleic acid and protein sequences with novel functions is also on the horizon. Only time will tell what new areas of biology may be influenced by the further development and application of this tech nique. Literature Cited 1. Saiki, R . , Scharr, S

. • Faloona, F. , Mul lis, K . B . • Hom, G. T. , Erlich, H . A . , Amheim, N. 1985. Science 230:1350-

8. Erlich, H. A . , Gelfand, D . , Sninsky, J . 1 99 1 . Science 252 : 1 643-5 1 9. Haff, L . , Atwood, J. G . , Dicesare, J . , Katz, E . , Picozza, E . , et al . 199 1 . Biotechniques 1 0 : 102- 1 2 1 0 . Chamberlain , J. S . , Gibbs, R . A . , Ranier, J. E . , Caskey, C . T . 1990. See Ref. 1 4 1 , pp. 272-8 1 1 1. Smith, K. T . , Long, C. M . , Bowman, B . , Manos, M. M. 1990. Amplifica tions, Sept: pp. 1 6- 1 7 . Perkin-Elmer

Gelfand, J . J . Sninsky, T . J . White, pp. 129-4 1 . New York: Academic 5 . Faloona, P . , Weiss, S . , Ferre, F. , Mul lis, K. 1990. 6th /nt. Conf. AIDS. Abstr. 6. D'Aquila, R. T. , Bechte l , L. J . , Videl er, J. A . , Eron, J. J . , Gorczyca, P. , Kaplan, J. C. 1 99 1 . Nucleic Acids Res.

1Z. Lubin, M . , Elashoff, J. D . , Wang, S-1 . , Rotter, J . , Toyoda, H . 1991. Mol. Cell. Probes 5 :307- 1 7 13. Syvanen, A-C. , Bengtstrom, M., Tenhunen, J . , Soderlund, H . 1 988. Nucleic Acids Res. 16: 1 1327-38 14. Chelly, J . , Kaplan, J. C . , Maire , P . , Gautron, S . , Kahn, A . 1988. Nature 333:858-60 15. Lawyer, F. c . , Stoffel, S . , Saiki, R. K . ,

54 2. Mullis, K. B . , Faloona, F. A. 1 987. Methods Enzymol. 155 :335-50 3. Saiki, R. K . , Gelfand , D. H . , Stoffel, S . , Scharr, S . , Higuchi, R . , et al. 1 988. Science 239:487-91 4. Gelfand, D. H . , White, T. J. 1990. In PCR Protocols, A Guide to Methods and Applications, ed. M. A. Innis, D . H .

1 9:3749 7. Li, H . , Cui, X . , Amheim, N. 1991. Proc. Narl. Acad. Sci. USA 87:4580-84

Corp .

154

ARNHEIM & ERLICH


Myambo, K . , Drummond , R . , Gelfand, D . H . 1989. J. Bioi. Chem. 264:6427-

37 16. Innis, M. A . , Myambo, K. B . , Gelfand, D . H . , Brow, M . A . D . 1988. Proc. Natl. Acad. Sci. USA 85:9436-40 17. Gelfand, D. H . 1 989. See Ref. 142, pp. 17-22 1 8 . Longley, M. 1 . , Bennett, S . E . , Mos baugh, D. W. 1990. Nucleic Acids Res. 18:7317-24 19. Tindal l , K. R. , Kunkel , T. A. 1988. Biochemistry 27 :6008-13 20. Holland, P. M . , Abramson, R. D . , Wat son, R . , Gelfand, D. H. 1991. Proc. Natl. Acad. Sci. USA 88:7276-80 2 1 . Clark, J. M. 1988. Nucleic Acids Res. 16:9677-86 22. Watson, R. 1989. Amplifications, May: pp. 5-6. Perkin-Elmer Corp. 23. Klimczak, L. 1 . , Grummt, F . , Burger, K. J. 1985. Nucleic Acids Res. 13:5269-

82

2 4 . Elie, c., Salhi, S . , Rossignol, 1-M . , Forterre, P. , deRecondo, A-M. 1 988.

Biochem. Biophys. Acta 95 1:261-67 25 . Salhi, S . , Elie, C . , Forterre , P. , de Recondo, A-M . , Rossignol , I-M. 1989. J. Mol. Bioi. 209:635-44 26. McConlogue. L . . Brow, M. A. D . , In nis, M. A. 1 988. Nucleic Acids Res. 1 6:9869 27. Myers, T . W Gelfand, D. H . 1991. Biochemistry 30:7661-66 28. Klimczak, L. J . , Grummt, F . , Burger, K. 1. 1986. Biochemistry 25:4850-55 29. Hamal, A . , Forterre , P . , Elie, C. 1990. Eur. J. Biochem. 190:517-21 30. Cariello, N . F . , Swenberg, J . A . , Skopek, T. R . 199 1 . Nucleic Acids Res. 19:4193-98 . •

3 1 . Goodenow, M . , Huet, T . , Saurin, W . , Kwok, S . , Sninsky, J . , Wain-Hobson, S. 1 989. J. Acquired lmmunol. Defic.

Syndr. 2 :344-52 32. Fucharoen, S . , Fucharoen , G. , Fucha roen, P . , Fukumaki, Y. 1 989. J. Biol. Chem. 264:7780--83 33. Mendelman, L. V . , Boosalis, M. S . , Petruska, I. , Goodman, M . F. 1990. J. Bioi. Chem. 264: 1441 5-23 34. Eckert, K. T . , Kunkel, T. A. 1990. Nucleic Acids Res. 18:3734-44 35 . Krawczak, M . , Reiss, J . , Schmidtke, I . , Rosier, U . 1989. Nucleic Acids Res. 17:2197-201 36. Paabo, S . , Irwin, D. M . , Wilson, A. C . 1990. J . Bioi. Chem. 265:4718-21 37. Meyerhans, A . , Vartanian, 1-P . , Wain Hobson, S. 1990. Nucleic Acids Res. 1 8 : 1687-91 38. Marton, A . , De1becchi, L . , Bourgaux, P. 1991. Nucleic Acids Res. 19:2423-26

39. Kwok, S . . Higuchi, R. 1989. Nature 239:237-38 40. Sarkar, G . , Sommer, S. 1990. Nature 343:27 4 1 . Longo, M . C . , Berninger, M . S . , Har ley, 1. L. 1990. Gene 93 : 1 25 42. Cimino, G. D . , Metchette , K. C . , Tess man, J. W . , Hearst, J. E . , Isaacs, S . 1991. Nucleic Acids Res. 19:99- 1 08 43 . Scharf, S . I . , Horn, G. T. , Erlich, H. A .

1986. Science 233: 1076--78 44. Aslanidis, c . . de Jong, P. J. 1 990. Nucleic Acids Res. 18:6069 45 . Silver, J . , Keerikatte , V. 1989. J. Cell. Biochem. Abstr. WH239, Supp\. 13E 46. Triglia, T . , Peterson , M. G . , Kemp, D. J . 1988. Nucleic Acids Res. 1 6:8186 47. Ochman, H . , Gerber, A. S . , Hartl, D. L . 1 988. Genetics 120:621-23 48. Mueller, P. R . , Wold, B. 1 989. Science 246: 7 80--86 49. Ko, M. S. H . , Ko, S. B. H . , Takahashi, N . , Abe, K . 1990. Nucleic Acids Res.

18:4293-94 50. Shyamala, V . , Ames, G. F. 1989. J. Cell. Biochem . SBE:306

5 1 . Ludeckc, H-J . , Senger, G . , Claussen, U . , Horsthemke , B. 1989. Nature

338:348-50

52. Wesley, C. S . , Ben, M . , Kreitman, M . , Hagag, N . , Eanes, W . F . 1 990. Nucleic

Acids Res. 18:599-603 53. Maniatis, T . , Fritsch, E. F. , Sambrook , J . 1982. Molecular Cloning, A Labora tory Manual. New York: Cold Spring Harbor Lab.

54. Todd, J . A . , Bell, J . I . , McDevitt, H . O . 1987. Nature 329:599-604 5 5 . Kawasaki, E. S . , Wang, A. M. 1989. See Ref. 142, pp. 89-97 56. Powell, L. M . , Wallis, S. C . , Pease, R . J . , Edwards, Y . H . , Knott, T . J . , Scott, I. 1987. Cell 50:831-40 57. Gilliland, G . , Perrin, S . , B lanchard, K . , Bunn, H . F. 1990. Proc. Natl. Acad.

Sci. USA 87:2725-29 58. Wang, A. M . , Doyle, M. V . , Mark, D . F . 1989. Proc. Natl. Acad. Sci. USA 86:97 1 7-2 1 59. Frohman, M. A . , Dush, M. K . , Martin, G. R. 1988. Proc. Natl. Acad. Sci. USA 85:8998-9002 60. Loh, E. Y . , Elliott, J. F . , Cwirla, S . , Lanier, L . L . , Davis, M . M . 1989. Sci ence 243:217-20 6 1 . O 'Hara, 0 . , Dorit, R. L . , Gilbert, W . 1989. Proc. Natl. Acad. Sci. USA 86:5673-77 62. Belyavsky, A . , Vinogradova, T . , Ra jewsky, K. 1989. Nucleic Acids Res. 1 7:29 1 9-32

63. Brunet, J-F . , Shapiro, E . , Foster, S. A . ,

peR STRATEGY Kandel, E. R . , lino, Y. 1991. Science


252:856-59

64. Welsh, J . , Liu, J-P. , Efstratiadis, A. 1990. Genet. Anal. Techn. Appl. 7:5-17 65 . Timblin, C . , Battey, J . , Kuehl, W . M . 1990. Nucleic Acids Res. 1 8 : 1587-93 66. Ko, M . S . H . 1990. Nucleic Acids Res. 18:5705-1 1 67. Patanjali, S . R . , Parimoo , S . , Weiss man, S. M . 1991. Proc. Natl. Acad. Sci. USA 88: 1943-47 68. Wieland, 1. , Bolger, G . , Asouline, G . , Wigler, M. 1990. Proc. Natl. Acad. Sci. USA 87:2720-24 69. Gould, S . J . , Subramani, S . , Scheffler, l. E. 1989. Proc. Natl. Acad. Sci. USA 86:1934-38 70. Kamb, A . , Weir, M . , Rudy, B . , Var mus, H . , Kenyon, C. 1989. Proc. Natl. Acad. Sci. USA 86:4372-76 7 1 . Libert, P . , Parmentier, M . , Lefort, A . , Dinsart, c. , Van Sande, 1 . , e t al. 1 989 . Science 244:569-72 72. Gautam, N . , Baetscher, M . , Aebersold, R . , Simon, M. I. 1989. Science 244:971-74 73. Wilks, A. F. 1989. Proc. Natl. Acad. Sci. USA 86:1603-7 74. Wilkie, T. M . , Simon, M . 1. 199 1 . See Ref. 143, pp. 32-41 75. Lee, C. c. , Wu, X . , Gibbs, R. A . , Cook, R . G . , Muzny, D . M . , Caskey, C. T. 1988. Science 239: 1288-91 76. Mack, D. H . , Sninsky, J. J. 1988. Proc.

Nat!. Acad. Sci. USA 85 :6977-81 77. Shih, A. , Misra, R . , Rush, M. G. 1989. J. Viral. 63:64-75 78. Nunberg, J. H . , Wright, D. K . , Cole,

G. E. , Petrovskis, E . A . , Pust, L. E . , et al. 1989. J. Viral. 63:3240-49 79. Greenberg, S . J . , Erlich, G. D . , Abbott, M. A . , Hurwitz, B . J . , Waldmann, T. A . , Poiesz, B. J. 1989. Proc. Natl.

Acad. Sci. USA 86:2878-82

80. Wu, D. Y . , Ugozzoli , L . , Pal, B. K . , Wallace, R . B . 1989. Proc. Natl. Acad. Sci. USA 86:2757-60 8 1 . Newton, C. R . , Graham, A . , Heptin

stal l , L. E . , Powel l , S. J . , Summers, C . , e t al. 1989. Nucleic Acids Res. 1 7 :2503-

16 82. Gibbs, R . A . , Nguyen, P-N . , Caskey, C. T. 1 989. Nucleic Acids Res. 1 7 :2437-48 83. Ehlen, T . , Dubeau, L. 1989. Biochem. Biophys. Res. Commun. 160:441-47 84. Petruska, 1 . , Goodman, M. F . , Boosa lis, M. S . , Sowers, L. c. , Chaejoon, c . , Tinoco, 1. 1988. Proc. Natl. Acad. Sci.

USA 85:6252-56 85. Kwok, S . , Kellogg, D. E . , McKinney, N . , Spasic, D . , Goda, L . , et al. 1990. Nucleic Acids Res. 18:999-1005

1 55

86. Cui, X . , Li, H . , Goradia, T. M . , Lange, K . , Kazazian, H. H. Jr., et al. 1989. Proc. Natl. Acad. Sci. USA 86:9389-93 87. Goradia, T. M . , Stanton, V. P. Jr. , Cui, X . , Aburatani , H . , Li, H. , et al. 1991 . Genomics 10:748-55 88. Amheim, N . , Li, H . , Cui, X. 1990. Genomics 8:415-19 89. McHeyzer-Williams, M. G . , Nussal, G. J . V . , Lalor, P. A . 1 99 1 . Nature 350:502-5 90. Rappolee, D. A . , Brenner, C. A . , Schultz, R . , Mark, D . , Werb, Z. 1988. Science 24 1 : 1823-25 9 1 . Rappolee, D. A . , Mark, D . , Banda, M . J . , Werb, Z. 1988. Science 24 1 :708-12 92. Chelly, J . , Concordet, J-P. , Kaplan, J C . , Kahn, A. 1989. Proc. Natl. Acad. Sci. USA 86:2617-21 93. Sarkar, G . , Sommer, S. S. 1989. Sci ence 244:331-34 94. Roberts, R. G . , B arby, T. F. M . , Man ners, E . , Bobrow, M . , Bentley , D. R .

199 1 . Am. J. Hum. GeneT. 49:298-3 1 0 95 . Sninsky , J . 1991. I n Viral Hepatitis and Liver Disease, ed. F. B . Hollinger, S .

M . Lemon, H . S . Margolis, pp. 799805. Baltimore: Williams and Wilkins 96. Wrischnik, L. A . , Higuchi, R . G . , Stoneking, M . , Erlich, H . A . , Amheim, N . , Wilson, A. C. 1987. Nucleic Acids

Res. 15(2):529-42 97. Stofiet, E. S . , Koeberl , D. D . , Sarkar, G . , Sommer, S. S. 1988. Science

239:49 1 -94

98 . Engelke, D. R . , Hoener, P. A . , Collins, F . S . 1988. Proc. Natl. Acad. Sci. USA 85:544-48 99. McMahon, G . , Davis, E . , Wogan, G . N . 1987. Proc. Natl. Acad. Sci. USA 84:494-78 100. Wong, C . , Dowling, C. E . , Saiki, R .

K . , Higuchi , R . G . , Erlich, H . A . , Kazazian, H . H . 1987. Nature 330:384--

86 1 0 1 . Gyllensten, U. B . , Erlich, H. A. 1988. Proc. Natl. Acad. Sci. USA 85:7652-56 102. Homes. E . , Hultman, T. , Moks, T . , Uhlen, M . 1990. Biotechniques 9:730 \03. Higuchi, R . , Ochman, H. 1989. Nucleic Acids Res. 1 7 :5865 \04. Nakamaye, K. L . , Gish, G. , Eckstein, F . , Vosberg, H. P. 1988. Nucleic Acids Res. 16:9947-59 105. Ruano, G. , Kidd, K. K. 1991. Proc. Natl. Acad. Sci. USA 88:2815 \05a. Lee, J-S. 199 1 . DNA Cell BioI. 1 0:6773 106. Gyllensten, U. B. 1989. See Ref. 142, pp. 45-60 107. Ephrussi, A . , Church, G. M . , Tonega wa, S . , Gilbert, W. 1985. Science 227: 134-40

156

ARNHEIM & ERLICH

1 08. Church, G. M . , Ephrussi, A . , Gilbert, W . , Toncgawa, S. 1985. Nature 3 1 3 :798-801 1 09. Pfeifer, G. P. , Riggs, A. D. 1 99 1 . Genes Dev. 5 : 1 102- 1 3 1 10. Pfeifer, G . P . , Steigerwald, S . D . ,


Mueller, P. R . , Wold, B . , Riggs, A . D .

1 989. Science 246:8 1 0-- 1 3 1 1 1 . Church, G . M . , Gilbert, W. 1984. Proc . Natl. Acad. Sci. USA 8 1 : 1 99 1 -95 1 1 2. Maxam, A. M . , Gilbert, W. 1 980. Methods Enzym ol . 65 :499-560 1 1 3 . Pfeifer, G. P . , Steigerwald, S . D . , Han sen, R. S . , Gartler, S. M . , Riggs, A. D .

1 990. Proc. Natl. Acad. 87:8252-56

Sci.

USA

1 1 4. Rideout, W. M . , Coetzee, G. A . , Olu mi, A. F . , Jones, P. A. 1 990. Science 249 : 1 288-90 1 1 5 . Pfeifer, G. P . , Drouin, R . , Riggs, A . D . , Holmquist, G . P. 1 99 1 . Proc. Natl. Acad. Sci. USA 88: 1 374-78 1 16 . Fischer, S. G . , Lerman, L. S. 1 983. Proc. Natl. Acad. Sci. USA 80: 1 579-83 117. Myers, R. M . , Maniatis, T. 1986. Cold Spring Harbor Symp . Quant. Bioi. 5 1 :275-84 1 18 . Keohavong, P . , Kat, A. G . , Cariello, N . F . , Thilly, W . G . 1988. DNA 7 ( 1 ) :6370 1 1 9. Sheffield, V. C . , Cox, D. R . , Lerman, L. S . , Myers, R. M. 1 989. Proc. Natl. Acad. Sci. USA 86:232-36 120. Wartell, R. M . , Hosseini, S. H . , Moran, e. J . Jr. 1 990. Nucleic Acids Res. 1 8 :2699-705 1 2 1 . Orita, M . , Iwahana, H . , Kanazawa, H . , Hayashi, K . , Sekiya, T . 1989. Proc. Natl. Acad. Sci. USA 86:2766--70 1 22. Orita, M . , Suzuki, Y . , Sekiya, T. , Hayashi, K. 1 989. Genomics 5:874-79 1 23 . Johnston, B . H . , Rich, A. 1 985. Cell 42: 7 1 3-24 1 24. McClellan, J. A . , Palecek, E . , Lilley, D. M . J. 1 986. Nucleic Acids Res. 14:9291-309 1 25 . Cotton, R. G. H . , Rodrigues, N. R . , Campbell, R . D . 1988. Proc. Natl. Acad. Sci. USA 85:4397-401 1 26. Montandon , A. J . , Green, P. M . , Gian nelli, F . , Bentley, D. R. 1 989. Nucleic Acids Res. 1 7:3347-58 127. Kinzler, K. W . , Vogelstein, B. 1 989. Nucleic Acids Res. 1 7 :3645-53 1 28 . Kinzler, K. W . , Vogelstein, B. 1 990. Mol. Cell. Bioi. 1 0:634-42 129. Galas, D. J . , Schmitz, A. 1978. Nucleic Acids Res. 5 : 3 1 57-70

1 30. Fried, M . , Crothers , D. M. 1 98 1 . Nucleic Acids Res. 9:6505-25 1 3 1 . Gamer, M. M . , Revzin , A. 1 98 1 . Nucleic Acids Res. 9:3047-60 1 32. Blackwell, T. K . , Weintraub, H . 1 990. Science 250: 1 1 04-10 1 3 3 . Thiesen, H-J . , Bach, C . 1 990. Nucleic Acids Res. 1 8 :3203-9 134. Mavrothalassitis, G . , Beal, G . , Papas, T. S. 1 990. DNA Cell Bioi. 9:783-88 1 35 . Tuerk, C . , Gold, L. 1 990. Science 249:505- 1 0 1 36. Ellington, A . D . , Szostak, J . W . 1 990. Nature 346: 8 1 8-22 1 37 . Green, R . , Ellington, A. D . , Bartel, D . P . , Szostak, J . W. 1 99 1 . See Ref. 143 , pp. 75-86 1 3 8 . Amheim, N . , Levenson, C. H. 1 990. Chem. Eng. News 68: 36-47 1 39. Amheim, N . , White, T. , Rainey , W . 1990. BioScience 40: 1 74-82 140. Erlich, H . A . 1 989. 1. CUn. lmmunol. 9:437-47 1 4 1 . Innis, M. A . , Gelfand , D. H . , Sninsky, J. J . , White, T. J . , eds. 1990. PCR Pro tocols, A Guide to Methods and Applica tions. New York: Academic. 482 pp. 142. Erlich , H . A . , ed. 1989. PCR Technolo gy: PrincipLes and Applicarionsfor DNA Amplification. New York: Stockton. 246 pp.

143. Amheim, N . , ed. 1 99 1 . Polymerase Chain Reaction. Methods: A Companion to Methods in Enzymology, Vol. 2. 86 pp.

144. Reiss, J . , Cooper, D. N. 1 990. Hum. Genet. 85: 1-8

145 . White, T., Madej , R . , Persing, D . H . 1 99 1 . Advances in Clinical Chemistry,

ed. H. E. Spiegel. In press 145a. White, T . , Amheim, N . , Erlich, H. A .

1989. Trends Genet. 5 : 1 85-89 146. Almoguera, e . , Shibata D . , Forrester, ,

147. 148. 149. 150.

K . , Martin, J . , Amheim, N . , Perucho, M . 1988. Cell 53:549-54 Shibata, D . , Martin, W. J . , Amheim, N. 1 988. Cancer Res. 48:4564-66 Smit, V. T. , Boot, A . J . , Smits, A . A . , Fleuren, G . J . , Comelisse, C . J . , Bos, J . L . 1 988. Nucleic Acids Res. 16:7773-82 Paabo, S . , Higuchi, R. G . , Wilson, A . C . 1989. 1 . BioI. Chern. 264:9709-1 2 Thomas, R. H . , Schaffner, W. , Wilson, A. e . , Paabo, S . 1989. Nature 340:465-

67 1 5 1 . Golenberg, E . M . , Giannasi, D. E . , Clegg, M . T . , Smiley, C. J Durbin M. et al. 1 990. Nature 344:656--5 8 . •

,

Quantitative polymerase chain reaction.

Inverse polymerase chain reaction.

Polymerase chain reaction techniques.

The polymerase chain reaction.

Polymerase chain reaction.

A rapid polymerase-chain-reaction-directed sequencing strategy using a thermostable DNA polymerase from Thermus flavus.

RNA template-specific polymerase chain reaction (RS-PCR): a novel strategy to reduce dramatically false positives.

DNA polymerase fidelity and the polymerase chain reaction.

Deletion mutagenesis during polymerase chain reaction: dependence on DNA polymerase.

Targeted gene walking polymerase chain reaction.

Polymerase Chain Reaction on a Viral Nanoparticle.

Genetic analysis using the polymerase chain reaction.

DNA amplification by the polymerase chain reaction.

Red light-controlled polymerase chain reaction.

Recent advances in the polymerase chain reaction.

[Gene amplification by the polymerase chain reaction].

Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase.

Polymerase chain reaction: applications in environmental microbiology.

The polymerase chain reaction (PCR): general methods.

Polymerase chain reaction: trenches to benches.

Genotyping errors with the polymerase chain reaction.

Heteroduplex formation in polymerase chain reaction.

Robust regression methods for real-time polymerase chain reaction.

Culture-negative endocarditis diagnosed using 16S DNA polymerase chain reaction.