Genetic analysis using the polymerase chain reaction.

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Annu.Rev.Genet. 1992. 26:479-506 Copyright © 1992 i1y Annual Reviews Inc. All

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GENETIC ANALYSIS USING THE POLYMERASE CHAIN Annu. Rev. Genet. 1992.26:479-506. Downloaded from www.annualreviews.org by NORTH CAROLINA STATE UNIVERSITY on 11/11/12. For personal use only.

REACTION Henry A. Erlich Department of Human Genetics, Roche Molecular Systems, Inc., 1145 Atla ntic Avenue, Alameda, California 94501

Norman Arnheim Department of Molecular Biology, University of Southern California, Los Angeles, California 90089-1340

KEY WORDS: genetic variation, linkage analysis, diagnosis, forensics, somatic mutations

CONTENTS INTRODUCTION .. .... . ......... . . .. ...................... The Reaction ....... ........... . ......... . ... ...........

480 480

METHODS FOR DETECTING GENETIC VARIATION IN AMPLIFIED DNA . . , Polymorphisms Due to Variation in Sequence (Unknown) . . . . ........ . . .

482 482

ADVANTAGES OF USING PeR FOR DNA-GENOTYPE DETERMINATION .. ,

Analysis When the Exact Nature of the DNA Sequence Differences Between Alleles is Known ....................................

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POLYMORPHISMS IDENTIFIED BY PeR ARE USEFUL FOR GENETIC MAPPING ........................................

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LINKAGE ANALYSIS BY PeR TYPING OF SINGLE GAMETES .... . ......

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PREIMPLANTATION GENETIC DISEASE DIAGNOSIS . .............. . .

ANALYSIS OF SOMATIC MUTATIONS USING PCR ...... . ...........

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DIAGNOSIS OF GENETIC DISEASE AND DISEASE SUSCEPTIBILITY ... ...

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TISSUE TYPING FOR TRANSPLANTATION . . . . . .. ..... ... . . .. . . ...

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MOLECULAR EVOLUTION . . . . . ........... . ..................

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FORENSICS . . . .

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SUMMARY ............................................. .

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INTRODUCTION Genetics has traditionally been a discipline that focussed on the analysis of inherited variation; these genetic variants served either as a means (as markers in genetic mapping or in population studies) or as an end (wild-type vs mutant

Annu. Rev. Genet. 1992.26:479-506. Downloaded from www.annualreviews.org by NORTH CAROLINA STATE UNIVERSITY on 11/11/12. For personal use only.

structure/function studies). The advent of molecular cloning and recombinant

DNA techniques such as Southern blotting allowed the analysis of DNA sequence variation and marked a new era in molecular genetics. The introduction of the polymerase chain reaction (PCR) (79, 94, 95), an in vitro method for the enzymatic amplification of specific DNA segments from a complex genome (1, 33, 117), has greatly simplified the study of sequence variation and, as a result, has transformed the way in which genetic analysis

can be carried out. PCR amplification of a specific DNA segment from genomic DNA or from RNA, following reverse transcription, can replace the construction of genomic and cDNA libraries for many applications and, in others, can greatly facilitate subsequent procedures, such as cloning, sequencing, oligonucleotide probe hybridization, or restriction site analysis. In addition to simplifying many procedures and, thus, allowing the use of molecular techniques in a wide variety of biological disciplines, PCR has made possible new approaches to genetic analysis based on the ability to amplify very small samples (e.g. sperm mapping; see below). In this paper, we review the use of PCR for molecular genetic studies. The Reaction PCR is based on the annealing and extension of two oligonucleotide primers that flank a specific "target" DNA segment; after denaturation of the duplex DNA, each primer hybridizes to one of the two separated strands such that the 3'OH ends are facing each other. The annealed primers are then extended on the template strands by a DNA polymerase. These three steps (denaturation, primer binding, and DNA synthesis) represent a single PCR cycle. If the newly synthesized strand extends through the region complementary to the other primer, it can then serve as a primer binding site and template for a subsequent primer extension reaction. Consequently, repeated cycles of denaturation, primer annealing, and primer extension generate an exponential accumulation of a discrete DNA fragment. This exponential amplification results because, under appropriate conditions, the primer extension products synthesized in cycle n function as templates for the other primer in cycle n + 1 . Since the initial PCR amplifications using DNA polymerase I Klenow fragment from E. coli (94) were reported, a variety of thermostable DNA polymerases, starting with Taq polymerase from Thermus aquaticus (95), have been introduced into the peR. The use of thermostable DNA polymerases eliminated the need for addition of fresh enzyme after the denaturation step in the thermal profile and, consequently, allowed the development of thermal


PCR AND GENETIC ANALYSIS

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cycling devices to automate the peR; the use of higher temperatures for primer annealing and extension has significantly increased the specificity (target vs nontarget product) of the peR. Recently, additional thermostable DNA polymerases isolated from the thermophilic eubacteria Thermus thermophilus (Tth) (79) as well as from the hyperthermophilic eubacteria, Thermotoga maratima (Tma) have been used in peR. Many of these DNA polymerases differ in terms of their thermoresistance, thermal activity profile, and processivity. Some enzymes (e.g. Taq polymerase) have a 5'-3' exonuclease but lack a 3'-5' exonuclease activity; some enzymes have both. Genetically engineered variants of these enzymes (e.g. the Stoffel fragment of Taq polymerase which lacks the 5'-3' exonuclease activity), have also been developed recently. Some of the recently isolated enzymes have properties that will be very valuable for particular applications. The polymerase from Thermus thermophilus (Tth) for example, has reverse transcriptase activity in the presence of manganese, allowing a single tube cDNA synthesis from an RNA template, followed by peR amplification of the cDNA, once the manganese has been replaced by magnesium (79). The ability to reverse transcribe at elevated temperatures under conditions of reduced secondary structure in the mRNA template should provide for efficient cDNA synthesis. Some polymerases may have increased fidelity as a consequence of 3'-5' (proofreading) exonuclease activity. The properties and applications in peR of various thermostable DNA polymerases have been reviewed recently (1). peR amplification of a specific DNA segment (e.g. -globin) uses two oligonucleotide primers flanking the region of interest; thus, classical peR amplification requires nucleotide sequence information for the design of the primers. This requirement for sequence information, however, can be over come using generic primers, such as oligonucleotides based on Alu sequences that allow amplification of a discrete set of DNA fragments bounded by inverted Alu sequences (see below). Similarly, the amplification of DNA segments unconstrained by a requirement for specific sequence information can be achieved by priming with random oligonucleotides (see below) or by ligating linkers onto genomic DNA fragments to serve as primer sites, as in genomic peR (59). With these approaches, the ability to amplify DNA is uncoupled from the selectivity of classical peR. In some cases, a specific DNA fragment can be selected by some other principle, like specific protein-DNA interaction, as in the identification of a p53-binding DNA sequence motif using genomic peR (28). ,

ADVANTAGES OF USING peR FOR DNA-GENOTYPE DETERMINATION There are a number of advantages to using a peR assay for determining the genotype of a DNA sample. The assay takes only a few hours compared to Southern blotting, which can take days or weeks. Secondly, peR is capable

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of being automated, allowing large numbers of samples to be rapidly typed with little labor required. Finally, PCR is economical of DNA; only picograms or nanograms are required, whereas nanograms or micrograms are needed for Southern blotting of genomic DNA. In one PCR reaction, many distinct genetic loci can be typed at the same time by multiplex amplification using multiple primer pairs. As a result of these economies, a number of laboratories have converted known RFLP markers into a PCR format and many searches for new polymorphisms have focused on those that can be assayed by PCR. METHODS FOR DETECTING GENETIC VARIATION IN AMPLIFIED DNA Polymorphisms Due to Variation in Sequence (Unknown) The most basic requirement for PCR analysis is a set of primers that flank a specific polymorphic DNA region. Once a specific PCR product has been amplified, various procedures can be used to determine the genotype of a sample. DIRECf DNA SEQUENCING The most complete characterization of a PCR product is the determination of the nucleotide sequence; this is the most sensitive way of revealing polymorphism. Although the sequence analysis of amplified DNA was initially performed on cloned PCR products (100), the increase in specificity of PCR made possible by the use of thermostable DNA polymerase has made direct sequencing of PCR products the preferred approach for most applications. Direct sequencing avoids potential problems associated with rare misincorporated bases (multiple clones must be sequenced to detect rare PCR artifacts such as misincorporation) but, in the amplification of multigene families or complex heterozygous samples, cloning is often useful to separate the different alleles or loci. Chain-termination sequencing is carried out with either labeled (radioactive or fluorescent) primers, with labeled dNTPs, or with labeled di-deoxy terminators. Single-stranded tem plates can be generated by using asymmetric PCR (45), an approach in which one of the two primers is limiting, or by using a biotinylated primer such that one strand of the PCR product can be captured by a streptavidin bead (56). The use of thermostable DNA polymerases has allowed the cycle sequencing approach in which repeated extension and denaturation cycles result in a linear accumulation of primer extension products increasing the sensitivity of the sequencing reaction (66, 93). Double-stranded templates can be sequenced readily since reassociation of the template strands is minimized. The avail ability of automated sequencing instruments promises that the sequence analysis of PCR products will be not only a critical research procedure but may, ultimately , be used in a clinical diagnostic context as well.



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RESTRICTION ENZYME DIGESTION A DNA segment containing an RFLP (a polymorphic restriction site) can be digested with a series of restriction enzymes until one is found that reveals a difference among individuals . As with genomic RFLP analysis, controls to ensure that the digestion is complete are necessary to minimize the possibility that a sample homozygous for the presence of a cutting site could appear to be a heterozygote. The formation of heteroduplex molecules in PCR products amplified from a heterozygote sample could prevent restriction enzyme digestion on a fraction of the PCR products . Not all alleles differ by nucleotide substitutions that cause a change at a restriction enzyme site. Thus, there are many more polymorphisms than can be detected by restriction enzyme analysis. Recently, a number of new technologies have been developed for determining whether a particular segment of DNA is polymorphic. While these methods can be used on cloned DNA fragments, it is clearly simpler to analyze PCR-amplified segments from different individuals rather than having to clone each gene fragment using recombinant DNA techniques .

Gel electrophoretic methods Denaturing gradient gel electrophoresis (DGGE) makes use of the property that two double-stranded DNA fragments that differ only by a single nucleotide substitution can have different thermal denaturation profiles. This difference can be detected by running the two DNA samples in adjacent lanes of an acrylamide gel that has a gradient of a DNA denaturant along the axis of DNA migration (37). 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. The fragment with the lower melting temperature will therefore migrate slower than the fragment with the higher Tm. Attaching a GC-rich stretch of nucleotides (clamp) to one of the PCR primers enhances the ability to detect single base substitutions ( 1 0 1 ) . DGGE is especially useful for mutation detection when large tracts o f DNA need to be examined for the specific position of a mutation in a gene. Two similar approaches to mutation detection have also been developed. One, thermal-gradient gel electrophoresis , makes use of a temperature as well as chemical gradient within the gel ( 1 1 2). A distinct approach to mutation detection by electrophoresis does not depend upon different melting temperatures in a denaturing gel but on differences in the secondary structure of single-stranded DNA. Subtle conformational differences due to the nucleotide sequence variation can be detected by running the PCR products made single stranded prior to electrophoresis on nondenaturing polyacrylamide gels (86, 87). Methods involving chemical or enzymatic mismatch cleavage Another ap proach to detecting point mutations is based upon the specific cleavage of


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ERLICH AND ARNHEIM

heteroduplex molecules formed when one nucleic acid strand is annealed to another strand that is complementary in sequence except for a single base. This base-pair mismatch can be detected by either chemical or enzymatic cleavage methods. In one chemical method, hydroxylamine chemically modifies mispaired Cs or osmium tetroxide chemically modifies mispaired Ts (20) . The modified base then is susceptible to cleavage by piperidine. Thus, a mismatch results in cutting of the DNA; the position of the cleavage site (or mismatch) can be determined by sizing the DNA fragments on a denaturing gel. This method has been applied recently to the analysis of mutations associated with phenylketonuria (38). Analysis When the Exact Nature of the DNA Sequence Differences Between Alleles is Known If the polymorphism at a locus has been characterized at the nucleotide sequence level, various simple and rapid methods can be used to study samples from different individuals. (a) One approach is to convert a polymorphism not detectable by restriction enzyme digestion to one that can be typed using a restriction enzyme (47) . This conversion is achieved by designing a PCR primer which, when incorporated into the PCR product , will introduce a mutation into one of the two alleles. The mutation makes the PCR product of the allele sensitive to enzyme digestion . This method can be used for rapid DNA typing of a large number of samples (H. H . Li & L. Hood, personal communication).

(b) Allele-specific probes Short labeled oligonucleotide hybridization probes can be used to distinguish single nucleotide differences in immobilized PCR products (the dot blot method) by ensuring through appropriately stringent hybridization and wash conditions that only a completely comple mentary probe will bind stably to the target sequence (96). This approach, termed PCRlSSO (sequence-specific oligonucleotide) typing, which extends earlier work with oligonucleotide probes on restriction digests of genomic DNA ( 1 7) , has been applied broadly to direct detection of disease mutations (e.g. sickle cell anemia , -thalassemia, cystic fibrosis) as well as to developing a general method of HLA typing (3 1, 32). PCR amplification of the target sequence allows the use of nonradioactive oligonucleotide probes and has made the PCRlSSO typing a general, simple, and robust diagnostic procedure. Since the typing procedure does not rely on the amplification of a unique diagnostic fragment, the use of a panel of probes to type multiple target sequences coamplified in a single reaction allows screening an individual sample for a variety of mutations and/or polymorphisms. For any genetic typing system, as the number of alleles and, hence the


485


number of labeled oligonucleotide probes increases, dot blot hybridization becomes more cumbersome. One approach to this problem has been the development of the "reverse dot blot" method (98) in which a panel of probes is immobilized and the immobilized array of probes is hybridized to a labeled PCR product in a single reaction. This method has been applied to HLA typing and p-thalassemia testing (32, 98) to forensics genetic testing (9 1) as well as to detection of specific mutations in the CFfR gene (Figure 1). (c) Allele-specific amplification The amplification reaction itself can also serve to distinguish sequence variants if the 3' end of one of the primers covers the variation. A primer mismatched at its 3' end with the template strand is much less efficiently extended by DNA polymerase (particularly those lacking the 3' -5' exonuclease activity) than a completely complementary primer. This approach, termed allele-specific amplification ( 1 20) or ARMS (amplification refractory mutation system (83), has been applied to sickle-cell anemia detection (120) as well as many other areas . Its power lies in the ability to detect very rare sequence variants (e.g. somatic mutations in tumor suppressor genes or oncogenes) in the presence of a huge excess of the normal allele. The nature of the mismatch at the 3' end of the primer affects the relative extension efficiency but judicious adjustment of the reaction conditions and thermal cycling conditions allows very sensitive (1110,000) detection of rare variants (J. Sninsky & M. Tada, personal communication). (d) Oligonucleotide ligation assay (OLA) A recently developed assay (63) capable of detecting single base differences is based upon the enzyme DNA ligase. In this procedure, an amplified sample is divided into two aliquots. Two 20-nucleotide long probe oligonucleotides that abut each another on the same strand are annealed to the denatured PCR product. One is allele-specific (capture probe) while the other serves as a reporter. If the allele-specific probe is perfectly complementary to the PCR product, then it can be ligated to the reporter probe. If the 3' end of the allele-specific probe is mismatched with the target at the polymorphic site then the ligation reaction will not occur. A biotin-labeled allele-specific capture probe and digoxigenin-Iabeled reporter probe have been used in this procedure. After the ligation reaction in the presence of PCR product, the biotin labeled allele-specific probe is captured on streptavidin-coated plates. The presence of digoxigenin in the captured material indicates sequence identity between the allele-specific probe and the PCR product, thereby allowing ligation of the two probes. The amount of digoxigenin can be quantitated by using alkaline phosphatase-labeled anti digoxigenin antibodies in an ELISA format. Automated methods have been developed and are based upon a 96-well microtiter plate ELISA-like assay


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6 different mutations in the CFfR gene. Biotinylated primer pairs specific for exons 1 0, 11, 20, and 21 were A through H. The presence of bound PCR product was revealed by incubating the strip with streptavidin-HRP and a chromogenic substrate. (A more detailed discussion is presented in Nersesian et a1; Reverse dot blot detection of

used to amplify a 492-bp, 425-bp, 391-bp, and 359-bp fragment, respectively, from DNA samples manuscript in preparation).


487


(84). The use of a thermostable DNA ligase from Thermus aquaticus has increased the specificity of this procedure (4).

(e) Length variation PCR primers that flank a region containing tandem repeats of either a simple sequence (e.g. di-, tri- or tetranucleotide repeats known as short tandem repeats) or a more complicated repeating unit allow examination of such length polymorphism on different chromosomes in the population by analyzing the length of the PCR products on agarose or acrylamide gels. This approach to genetic typing holds promise for a number of purposes, particularly genetic mapping. For example, as many as 50,000 loci in the human genome may contain (CA)n repeats and a significant fraction of these may be polymorphic (73, 113). The poly A tracts at the 3' end of transposed Alu sequences also appear to be polymorphic and could provide another rich source of genetic markers based on length polymorphism since there are almost one million Alu sequences in the human genome (25). The so-called minisatellite family of sequences discovered by Jeffreys as well as a host of additional VNTR region sequences also provide significant length polymorphisms for human genome analysis that can be analyzed by PCR (see Forensics section). POLYMORPHISMS IDENTIFIED BY PCR ARE USEFUL FOR GENETIC MAPPING The construction of genetic maps of DNA polymorphisms has relied on the analysis of RFLPs and length polymorphisms, including microsatellite repeats. Single nucleotide substitutions are also useful for such purposes. In addition to the methods described above for polymorphism detection, two new sources of polymorphism have been recently discovered and are based on a novel PCR strategy . In one strategy , a single PCR primer with an arbitrary sequence is used to amplify genomic DNA (114, 118). Amplification with this single primer under conditions of reduced stringency can give rise to a finite number of DNA fragments identified by gel electrophoresis. The origin of these fragments is unknown but presumably they contain genomic DNA sequences similar to the primer sequence that are relatively close together but in an inverted repeat orientation. When the fragment pattern is reproducible and differs between strains of mice or other individual organisms the different fragments can be analyzed for linkage ( 1 15, 1 16). This approach has also been given the acronym RAPD (Random Amplified Polymorphic DNA) and has also been useful for mapping in plant species. Repetitive elements are interspersed throughout the chromosomes of most eukaryotic organisms. If a single PCR primer composed of a consensus SINE (alu) or LINE (Ll ) repeat sequence is added to a sample containing human ,


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ERLICH AND ARNHEIM

DNA and amplification is carried out, a number of discrete DNA products are produced (81). These interspersed repetitive PCR products are composed of DNA segments that lie between two adjacent repeats that have an inverted orientation with respect to one another. Any specific chromosomal segment would be expected to have its own characteristic arrangement of repeated elements that would produce its own characteristic fragment pattern or fingerprint. This property has been used in the physical mapping of cosmid, YAC, or somatic cell hybrid DNA segments. Genetic mapping using interspersed repetitive PCR products is also possible. The patterns of fragments produced by PCR using mouse LINE and/or SINE consensus sequences differ between the closely related species Mus domesticus and Mus spretus. Since the mice can be crossed to one another, the genetic segregation pattern of the observed fragments and the linkage relationship among them were studied (22, 57). LINKAGE ANALYSIS BY peR TYPING OF SINGLE GAMETES Measuring the frequency of recombination between genetic markers is ultimately a question of determining the fraction of meiotic products that have undergone a genetic exchange. In humans, only those meiotic products that contribute to the formation of a diploid individual can be studied. Tradition ally, the frequency of recombination between human genetic markers has been calculated by studying pedigrees from informative families. This traditional approach deduces the genotype of the meiotic products involved in a fertilization event by analyzing the somatic cells of the progeny. A new approach based on PCR has been proposed to measure genetic recombination between DNA polymorphisms in higher organisms (69). The method, called sperm typing, analyzes the genotype of each meiotic product directly and does not require the study of diploid offspring. Instead, polymorphic segments from linked loci in each individual male meiotic product are coamplified by PCR. Recombination frequencies between DNA polymorphisms can be measured in this way without the traditional genetic cross or pedigree analysis. The principle of sperm typing is shown in Figures 2 and 3. An individual heterozygous for a microsatellite repeat at each of two linked loci is shown in Figure 2. This man will produce four kinds of sperm; two nonrecombinant types and two recombinants. Using PCR primers made from DNA sequence information flanking each polymorphic stretch of DNA, the two loci are coamplified simultaneously. The difference in the length of the PCR products at each locus is indicative of the number of microsatellite repeats characteristic of each allele. In this case the genotype of each sperm, whether it is a



489

recombinant or nonrecombinant, can be determined directly by gel electro phoresis. Using the methods described earlier in this chapter, the genotype of a single sperm can of course be determined even where the allelic difference at a locus is due simply to a single base substitution. Once a sufficient number of individual sperm has been typed, the recombination fraction between DNA polymorphisms is then estimated. In principle, the recombination fraction can be calculated by dividing the number of recombinant sperm by the total number examined. The data generated by the sperm-typing procedure is subject to experimental error: one or more of the regions being studied may not be efficiently amplified; the sample may be contaminated, the sample tube may contain more than a single sperm; or the sperm may contain more than a single homolog (aneuploid). A maximum likelihood approach has been developed to analyze sperm typing data that are potentially complicated by these factors (24). This method estimates these errors while at the same time calculating the recombination fraction and its standard error. Sperm typing accurately measures recombination fractions (24) even with rare DNA polymorphisms (55) and determines map order in three point crosses (43). In addition to the studies on human sperm, both sperm (67) and oocytes (23) from cows and mice, respectively, have been used to measure genetic recombination. To make use of many of the potential advantages of mapping DNA polymorphisms by sperm typing, large numbers of sperm need to be analyzed quickly and efficiently. Single sperm cells have been isolated by fluorescent activated cell sorting (FACS) so that a single sperm is automati cally placed into each well of a 96-well microtiter dish. Sperm isolation can be directly coupled with amplification (71) by employing a PCR machine that can use the same 96-well microtiter dish. In a recent study, a single individual analyzed 2000 sperm in a month (Karin Schmitt, personal communication). The capacity for studying recombination at such a high resolution is beyond the practical limits of family studies. Sperm typing will be especially useful for studying the relationship between physical distances and genetic recom bination fractions and for identifying recombination hot spots in male meiosis. It will also be valuable for examining potential differences between individuals for recombination frequency in the same interval. In principle, sperm typing can be applied to mapping DNA polymorphisms in any species where haploid gametes can be obtained, including microorganisms, plants (pollen typing), and animals. Of course, only male meiosis can be studied but gene order is the same in both sexes. Sperm typing allows only three loci to be studied simultaneously with reasonable efficiency; once a sperm has been analyzed in this way, it cannot be studied again. This property has precluded the use of sperm typing to make genetic maps in an efficient way. The development of a new procedure, Primer Extension Preamplification (PEP) (121), removes this obstacle. Using PEP,

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Figure 2 Male meiotic products from an individual doubly heterozygous for microsatellite CA repeat polymorphisms. The four chromatids characteristic of the first meiotic division are shown. At each locus the individual is heterozygous with respect to the number of repeats. Four types of sperm are expected to be produced.

at least 30 copies of all of the DNA sequences originally present in the single cell can be produced before individual DNA polymorphisms are examined, allowing many different polymorphisms to be analyzed from each peR. This method, in combination with the demonstration that highly polymorphic microsatellite repeats can be typed in single cells (55), will make it possible to use sperm typing for multipoint genetic mapping. By analogy to pedigree analysis, the sperm donor is the father who produces offspring (sperm) without the need for a maternal contribution (Figure 4). Large numbers of fathers are available and each father is capable of having a virtually unlimited number of progeny. Using PEP, at least 20, and perhaps as many as hundreds of different loci can be examined in each sperm. Traditional multipoint mapping statistical programs can be used to analyze the sperm-typing data


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Figure 3 PCR analysis of one individual sperm cell. Two sets of PeR primers (solid and filled rectangles) that directly flank the polymorphic sites are used to amplify the two loci. Electrophoresis of the PCR products is expected to reveal the genotype of the sperm. Shown in the lower part of the figure are the electrophoretic profiles expected from the diploid sperm donor and the four types of haploid sperm.

(88) by incorporating the assumption that the mother is never informative for any polymorphism. PREIMPLANTATION GENETIC DISEASE DIAGNOSIS The ability to study DNA sequences in a single haploid or diploid cell led to the idea that genetic disease diagnosis could be carried out in a human embryo produced by in vitro fertilization prior to implantation (21, 48, 69) . Handyside et al (48) showed that single blatomeres can be removed from human embryos at the 6- or 8-cell stage and highly repeated Y chromosome sequences can be detected by peR. In their report, a large fraction of the sampled human embryos developed into later stages in vitro, which suggested the possibility

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Figure 4 Comparison of the sperm.typing approach to measuring recombination with that of classical pedigree analysis. In the former an unlimited number of sperm donors and sperm can be studied but at the present time only 20-30 different loci can be studied in any one gamete.

Using the DNA from children in families allows a virtually unlimited number of loci to be studied but only a relatively small number of families such as those in the Centre pour les Etudes de Polymorphisme Humaine and Venezuelan pedigree are available.

that the diagnosed embryos could be implanted in the mother's uterus for further development. Recently, pregnancies (49) from biopsied embryos sexed by Y chromosome·specific DNA amplification have been reported and normal births resulted. Studies similar to these have also been carried out in mice (9, 42, 52). Preimplantation genetic disease diagnosis could be made even before fertilization by analyzing DNA sequences in the first polar body accompanying the oocyte (76, 106) . If the polar body from a woman heterozygous for a disease gene is found to contain the mutant allele, then the oocyte can be assumed to contain the normal allele an\l thus fertilized and implanted. The experimental error that can be associated with PCR make it necessary to provide some estimate of the risks associated with preimplantation genetic disease diagnosis. Also, recombination between the centromere and DNA marker must be considered. In general, it would be prudent for the clinical laboratory engaged in such a program to carry out extensive preclinical trials (77). Mathematical analyses of the risk of making diagnostic errors as a function of PCR efficiency, single cell manipulation and contamination have been made (80). The PEP procedure mentioned above would make it possible to conftrm the diagnosis on additional aliquots from the sample.


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ANALYSIS OF SOMATIC MUTATIONS USING PCR Methods of mutation analysis have been rooted in phenotypic selection. Cells that survive a treatment as a result of their containing a newly arisen mutation can be counted and the mutation rate calculated. The development of PCR has made it possible to conceive of measuring mutation rates without regard to phenotype. Genotypic selection (18) is based upon selectively amplifying a single mutant molecule in a background of unmutated DNA. Consider a sample of one million di loid cells. The specific amplification of a normal p unique sequence gene 10 bp in size is routine when starting with a genome with the complexity of 109 bp. The target is at a concentration of 10-6 and there are two million copies of the target in the sample. If one of the million cells was homozygous for a mutation and only the mutant copies of the gene were the target of amplification then the concentration of mutant template is 10-12 and consequently very difficult to detect. Several attempts have been made to detect specific mutations in a pool of normal genomes. Elhen & Dubeau used a method for allele-specific PCR (see above) in which efficiency was sacrificed for increased specificity and presented data su esting that one ¥ mutated ras oncogene could be amplified in the presence of 10 normal copies. However, additional confirmation of their approach is needed (26). A s noted above, Tada & Sninsky (personal communication) have also been able to detect very rare sequence variants by judicious manipulation of PCR param eters and comparisons of various mismatches. In another study, Cortopassi & Amheim (19) showed that by using the appropriate PCR conditions, mitochondrial genomes with a large deletion could be detected in the presence of several thousand normal genomes and recent unpublished studies show that even one in a million are detectable. It seems possible that highly specific PCR strategies will allow the detection of some mutant molecules at low concentration in a pool of normal genomes. This approach should prove ueful in· a variety of applications, including clinical detection of rare somatic mutations in. tumor-suppressor genes or oncogenes. DIAGNOSIS OF GENETIC DISEASE AND DISEASE SUSCEPTIBILITY The first PCR amplification of genomic DNA was of a 110-bp -globin fragment for the diagnosis of sickle-cell anemia (30, 94). Since these initial reports using the method of oligomer restriction to distinguish the �A and �s alleles, a variety of PCR-based methods, including allele-specific oligonucle otide (ASO) probe hybridization (96), restriction digestion (78), and allele specific amplification (120) have been applied to sickle cell anemia (SCA) diagnosis. Carrier detection and prenatal diagnosis for diseases resulting from point mutations or small insertion or deletions such as �-thalassemia (95),


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phenylkenoturia (27), cystic fibrosis (16, 33), Tay-Sachs, Gaucher's Disease (6) as well as many other less frequent diseases, have been accomplished by detection of specific mutations . In general, diagnosis based on direct detection of mutations requires an extensive knowledge of the spectrum of mutations at the disease locus. If only a small proportion of the mutations are known, linkage analysis in pedigrees °with an affected member can still be carried out, although an effective carrier screening program depends on identifying most of the mutations . In addition to the analysis of point mutations, PCR has facilitated the diagnosis of Duchenne's Muscular Dystrophy (DMD), an X-linked disorder, by detecting large deletions in the dystrophin gene (5, 39). Multiplex amplification of as many as nine exons in a single reaction is used to screen male samples to identify missing exons, hence, deletions . Careful quantitation of the specific PCR products, enhanced by the use of fluor escent primers and the ABI Genescanner, may allow identification of carrier females. The molecular analysis of another X-linked disorder, the Fragile-X syn drome, has revealed a novel genetic mechanism for this disease, which is characterized by mental retardation, and allowed the development of PCR based diagnostic tests. In this syndrome, a length polymorphism involving a CGG repeat at the 5' end of the FRX gene (14, 91), can give rise to an increased length variant (the pre-mutation) that has no clinical phenotype but is genetically unstable and can generate longer CGG repeat variants (muta tions) associated with the Fragile-X phenotype. PCR amplification with primers flanking the CGG repeat can distinguish the normal length range of polymorphic variants from the longer pre-mutation variants; however, some of the unstable mutation variants are sufficiently long that PCR amplification of the CGG repeat is inefficient and Southern blotting may be required for detection. Several other diseases, including Myotonic Dystrophy (MD) and Kennedy's Disease (also known as spinal bulbar muscular atrophy), have recently been shown to involve a similarly unstable trinucleotide repeat region (14, 91). The increase in repeat region size appears to account for the phenomenon of "anticipation" in which the clinical severity of the MD mutation increases from generation to generation. Common diseases with a strong component of genetic predisposition, such as cancer, cardiovascular disease, and autoimune disorders, represent another medical diagnostic area where analysis by PCR has proved informative. For neoplastic diseases such as breast cancer and col . reveal the inheritance of germline mutations in the p53 gene (for the breast cancer associated with the Li-Fraumeni syndrome, 74) and in the APC gene (for Familial Adenomatous Polyposis; 85). Detection by PCR of somatic mutations in these and in other tumor-suppressor genes may prove valuable



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in early detection, prognosis, and monitoring of residual disease following therapy. In cardiovascular disease, mutations in the LDL receptor gene, in the ApoB gene, as well as polymorphic variants in the ApoE and Apo(a) loci can all contribute to increased risk. Most of these variants can be detected by PCR (53, 62, 73, 108). It is in the analysis of the genetic predisposition to specific autoimmune diseases, however, that PCR has made the largest contribution. Many autoimmune diseases, such as insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, multiple sclerosis, celiac disease, and Pemphigus vulgaris, are positively (increased risk) or negatively (protection) associated with specific alleles of the HLA class II (DR, DQ, and DP) loci. For all of these diseases, PCR-based HLA typing, which is much more informative than blood-cell typing using serologic reagents (i.e. it distinguishes more alleles), has shown that specific alleles or, in some cases, combinations of alleles, confer increased risk (83). Moreover, PCR-based HLA typing has revealed the critical importance of individual polymorphic residues in the DRI3, DQI3, and DPI3 chains and, in the case of IDDM, demonstrated that specific alleles at both the DRB 1 and DQB 1 loci can contribute to susceptibility as well as to protection (35). One simple but important practical benefit of PCR-HLA typing is that noninvasive samples such as plucked hairs or cheek swabs can be analyzed instead of the conventional blood sample. Figure 5 shows a pedigree with three IDDM sibs, analyzed from hairs and swabs sent from family members to the lab in the mail. TISSUE TYPING FOR TRANSPLANTATION The same advantages of PCR-HLA typing discussed above with respect to disease susceptibility also apply to the typing of donor and recipients for solid organ and bone marrow transplantation. PCR-HLA typing, particularly in the reverse dot blot format mentioned above, is faster, simpler, and much more discriminating than conventional serologic typing. Recently, in a successful attempt to find an HLA-matched sib to serve as a bone marrow donor, we performed HLA PCRlSSO typing on hairs sent in the mail from family members of the patient. Since the number of alleles distinguishable by PCRlSSO typing is so much greater than the serologic types, more precise typing promises to increase the success rate of unrelated bone marrow donor transplants but also will increase the number of donors to be screened before a "match" can be found. MOLECULAR EVOLUTION Molecular approaches to the evolution of species and of populations have relied on the analysis of variable characteristics of specific proteins (e. g .

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A: ORB1-0402, OQAI-0301, OQBI-0302


B:

ORB1-1302, OQAI-0102, OQBI*0604/5

B/C 100M onset age 36

AIC 100M onset age 31

c:

D:

ORB1-0405, OQAI-0301, OQBI-0302 DRB1*0403/6, OQAl*0301. OQBI-0302

AIC

100M onset age 8

Figure 5 PCR HLA class II typing of hair and cheek swab samples from a family with three IDDM sibs. Closed boxes represent patients with IDDM. DRBl, DQAl and DQBl typing was carried out using PCR amplification snd hybridization with HRP-labeled oligonucleotide probes

as described (12,32,99). The DR4 subtypes segregating in this U.S. Caucasian multiplex IDDM family, DRB I *0402 and DRB I *0405 are both high risk DR4 haplotypes (35) that are rare in the

U.S. Caucasian population.

electrophoretic mobility and immunologic cross-reactivity) or on the restric tion endonuclease cleavage patterns or the nucleotide sequences of specific genes. In general, the nucleotide sequence in DNA provides the most informative set of molecular characters for the reconstruction of the evolu tionary history of individuals and of species. Computer programs have been developed for generating phylogenetic trees based on the nucleotide sequences

of a given gene from different individuals in various contemporary species. Prior to the introduction of peR, the difficulty in obtaining so much sequence information prevented the approach of molecular systematics, based on the construction of gene trees, from realizing its full potential . The analysis of population trees, based on differences in allele frequencies among populations, has also benefitted significantly from the use of peR-based genetic markers. Although the use of genomic RFLP markers for the study of DNA polymor phism represented an important advance over studies of protein markers (e. g. allozymes) in popUlation analysis, these studies were still time-consuming and required large amounts of DNA, generally requiring the establishment of immortalized cell lines from blood samples of the population. peR amplifi cation followed by a simple genetic typing procedure (e.g. oligonucleotide ,



497

probe hybridization) has already generated a significant body of data on the distribution of HLA alleles in various human populations (29). Using primers complementary to conserved DNA sequences, the ability to analyze DNA segments amplified from the nuclear as well as the mitochondrial genome has generated highly informative data sets for phylogenetic analysis . Recently, PCR has been used to obtain mitochondrial DNA sequences from the polymorphic D-Ioop region from a variety of individuals from different human populations as well as from nonhuman primates (61, 110); the phylogenetic tree constructed from these data suggests an African origin for modem humans and that all contemporary mitochondrial DNA lineages can be traced back to a woman (Mitochondrial Eve) who lived about 100-200,000 years ago, consistent with earlier studies based on mitochondrial DNA-re striction maps (13). Although recent statistical analysis has challenged the robustness (statistical confidence) of some aspects of these mitochondrial DNA phylogenies (50, 109), the interpretation of an African origin is very likely correct because it is consistent with phylogenetic trees based on nuclear gene frequencies (8) and with the observations that Africans display more genetic diversity (both in terms of mitochondrial lineages and nuclear gene alleles) than other human populations. (This argument is based on the assumption that the ancestral, hence oldest, human group should have had the most time to accumulate genetic variants in the population.) PCR has also facilitated the phylogenetic analysis of the origins of polymorphism in the major histocompatibility complex (MHC) by using primers to conserved regions of the human HLA class II loci to amplify polymorphic DNA segments from a wide range of contemporary primate species (45, 46). These and other studies of MHC evolution (36, 64) indicate that many of the modem MHC allelic lineages were present in the ancestral species prior to divergence of the hominoids (34, 60). Given certain assumptions about selection pressures, the size of the founding human population (about 10,000) can be estimated from these studies of ancestral MHC polymorphism (107). The argument that the results of the MHC phylogenetic analysis are incompatible with the Mitochondrial Eve hypothesis (60) does not take into account the stochastic aspect of matrilineal inheritance. Similar estimates (N 6,000 for human females) for the founding human population size have been derived from analyzing the sequence diversity in mitochondrial DNA (119). Nucleotide sequences that evolve slowly, like portions of the ribosomal RNA coding sequences, are valuable for phylogenetic analysis of widely divergent species. PCR has been used to generate ribosomal DNA sequences from a variety of fungal species to resolve the phylogenies of the fungi (11). Bacterial phylogenies have also been examined using primers based on conserved 16S ribosomal sequences (10). In an elegant approach to identifying =


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new microbial species, termed "characterizing the uncultured" (103) con served ribosomal primers were used to amplify 16S ribosomal DNA sequences from seawater samples from the Sargasso Sea (40). Following cloning and sequencing of the PCR products, 16S ribosomal DNA sequences from known aquatic microorganisms were identified, as well as sequences representing potential new species. These new ribosomal sequences can be analyzed phylogenetically so that the evolutionary relationship of these potential new species to known species could be established. This dramatic result illustrates the power of PCR using conserved region primers to detect new sequences. . These studies were all carried out with samples from extant species. The capacity of PCR to amplify small amounts of degraded DNA, however, has for the first time allowed the sequence analysis of ancient DNA. Samples from extinct species, such as the wooly mammoth and the quagga, a zebra-like animal, have been subjected to PCR amplification for tRNA genes (Higuchi, personal communication; 89). MHC sequences from a 7 ,000-year old mum mified human brain have also been amplified and sequenced (65) as has mitochondrial DNA from 600-year old human bones (M. Stoneking, personal communication). In general, museum specimens, such as the dried skin of the quagga or the kangaroo rat, will provide a rich source of samples for sequencing and for phylogenetic analysis. The current record for the PCR product from the oldest template belongs to the group that amplified sequences from a 17-20 million year old fossil leaf, related to contemporary magnolia (41). Although there is some controversy regarding potential contamination associated with this result, the PCR product from another species in the same fossil site has recently been reported (104). The current ease of obtaining PCR-based sequence information and the ability to amplify ancient DNA has already had a significant impact on evolutionary studies, with museums of natural history re-examining their collections in light of the potential of PCR amplification. PCR can also play a critical role in the analysis of human museum specimens, like the bloodstains and bone fragments from Abraham Lincoln's autopsy, to help answer historical questions, such as whether or not Lincoln had Marfan's Syndrome (75). FORENSICS As noted above, the use of PCR has illuminated our evolutionary past by facilitating the reconstruction of phylogenies based on DNA sequence. In a forensics setting, PCR has also allowed the reconstruction of more recent historical events, such as murder or rape, by analyzing the genetic pattern of polymorphic DNA sequences amplified from biological evidence found at the crime scene. Conventional forensics genetic markers, like the blood group serologic types or protein electrophoretic variants, cannot be applied to many



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forms of forensic evidence. In addition, the use of DNA fingerprinting, the analysis of genomic length polymorphisms (variable number tandem repeats or VNTRs) based on Southern blotting, typically requires reasonably large amounts (> 100 ng) of intact, high molecular weight DNA. peR is capable of analyzing minute quantities of degraded DNA. One peR-based test, a reverse dot blot test for polymorphism at the HLA-DQa (DQA l ) locus (HLA-DQa Amplitype ™ kit), has been applied by many forensics labora tories to the analysis of over 400 different cases since the first case (Pennsylvania vs Pestinikis) in 1986 (7) . For most of these evidence samples (typically >5 evidence specimens/case), DNA typing based on RFLP analysis could not provide a genotyping result. In general, peR typing of evidence samples for individual identification can be based either on the analysis of length polymorphism (VNTR regions or di, tri, or tetranucleotide repeats, known as short tandem repeats, STR, regions) or sequence polymorphism (see above). The amplification of VNTR or STR regions can be carried out by using primers complementary to unique sequences that flank the tandem repeat regions and allelic variants can be distinguished by determining the length of the peR products amplified from the sample. However, only a subset of the VNTR markers that have been analyzed by RFLP typing can be easily converted to peR markers, namely those in which the longest allele is less than 3 kb. In the past, peR products substantially longer than 3 kb have not been amplified efficiently from genomic DNA. In addition, if the two alleles determine peR products of very different lengths, the smaller fragment can, under conditions of limiting DNA polymerase, be preferentially amplified (111). The analysis of degraded DNA can also pose problems in that the larger allelic fragment is preferentially lost when amplifying a VNTR polymorphism from a partially degraded DNA sample. The analysis of STR regions does not appear to share with VNTR region amplification the same potential problems associated with preferential amplification and DNA degradation. For both VNTR and STR region markers, the definition of alleles in the sample is made using an allelic size ladder, a pool of peR products comprising the range of allelic size variants in the population. In some cases, however, sequence polymorphism may also be present in VNTR regions, making the definition of allelic length variants by electrophoretic mobility difficult. One recent approach to peR-based genetic typing, the MVR (minisatellite variable repeat) approach (58), utilizes both the length and sequence polymorphism in some VNTR regions for purposes of individualization. Sequence polymorphisms can be detected by a variety of methods (see above); in our experience, the use of oligonucleotide hybridization probes represents a general, robust, and powerful approach to genetic typing. With the use of the reverse dot blot method (98), in which a panel of oligonucleotide


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probes is immobilized in a spatial array on a nylon membrane, the complexity of the test is independent of the number of probes because the amplified sample is analyzed with all of the probes in a single hybridization reaction. This format has been developed for typing the polymorphism at the HLA DQAl (previously DQu) locus and is commercially available as the first PCR-based genetic typing kit. If the genotype of the evidence sample differs from that of the suspect' s reference sample, the suspect is excluded or eliminated as a potential donor of the evidence. If the genotypes of the suspect and evidence sample match, the suspect is included, that is, cannot be eliminated as the donor. The significance of this match for identifying the source of the evidence specimen is a function of the frequency of this specific genotype, as determined by population surveys (5 1 ) . Based on the available data on the distribution of HLA-DQu alleles, the probability that two individuals in a population will be indistinguishable (Pi) is 0.06 for Cauca sians. In the >200 cases performed by Forensic Science Associates in which the HLA-DQu test has yielded conclusive results, 65% have resulted in an inclusion while 35% have resulted in an exclusion (7) . Similar dot blot tests for determining the sequence polymorphism in the D-Ioop segment of mitochondrial DNA have recently been developed and applied to the forensic analysis of bone fragments ( 1 05). Recently, a genetic test has been developed (90) that incorporates the coamplification of five additional marker loci (each of which has two or three alleles), HLA-DQu, and analysis of the PCR products by the reverse dot blot method. This test, known as the Polymarker Kit, will be the second commercially available PCR test for human identifi cation and will provide a combined Pi (the probability that two random individuals will have the same genotype at these loci) of

The polymerase chain reaction.

Identification of Brucella spp. by using the polymerase chain reaction.

Diagnosis of haemophilia B using the polymerase chain reaction.

Detection of Listeria monocytogenes by using the polymerase chain reaction.

Detection of Helicobacter pylori by using the polymerase chain reaction.

Quantification of human cytomegalovirus DNA using the polymerase chain reaction.

Quantitative polymerase chain reaction.

Inverse polymerase chain reaction.

Polymerase chain reaction techniques.

Detection of Borrelia burgdorferi using the polymerase chain reaction.

Polymerase chain reaction strategy.

Strain identification of Actinobacillus actinomycetemcomitans using the polymerase chain reaction.

Polymerase chain reaction.

Applications of the polymerase chain reaction to genome analysis.

Analysis of the 14;18 translocation in cutaneous lymphomas using the polymerase chain reaction.

Molecular-genetic analysis of ocular adnexal benign lymphoid hyperplasias by a two-step polymerase-chain-reaction.

Mitochondrial DNA mutations associated with neuromuscular diseases: analysis and diagnosis using the polymerase chain reaction.

Analysis of transgenic animal pedigrees using the junction-polymerase chain reaction.

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

A general and rapid mutagenesis method using polymerase chain reaction.

Sensitive detection of Helicobacter pylori by using polymerase chain reaction.

Basic quantitative polymerase chain reaction using real-time fluorescence measurements.

DNA amplification by the polymerase chain reaction.

Diagnostic RAS mutation analysis by polymerase chain reaction (PCR).