journal of lnternal Medicine 1991: 230: 391-395
Review article The polymerase chain reaction and its potential role in clinical diagnostics and research J. REISS From the Institut f u r Hurnangenetik der Universitat. Giittingen. Germany
Abstract. Reiss J (Institut fur Humangenetik der Universitat, Gottingen. Germany). The polymerase Ehain reaction and its potential role in clinical diagnostics and research. journal of Internal Medicine 1991: 230: 391-395.
In-vitro amplification of deoxyribonucleic acid molecules by means of the polymerase chain reaction (PCR) must be regarded as the most important advance in the life sciences to take place during the last decade. Originally applied to the identification of mutations in well-known and fully sequenced human genes, its applications have now been extended to a wide variety of biological and medical disciplines, accompanied by significant technical improvements and sophisticated variations of the basic principle. Specialized molecular genetics laboratories were the first to employ this new method, and they still are in the process of extending its potential. Due to its unique properties, applications of PCR quickly spread to other areas of research, and numerous clinical studies have already employed PCR. The field is currently still expanding rapidly. Keywords : clinical research, diagnostics, DNA, polymerase chain reaction.
Introduction ‘The D N A duplex would be denatured to form single strands. This denaturation step would be carried out in the presence of a sujjiciently large excess of the two appropriate primers. Upon cooling, one would hope to obtain two structures, each containing the full length of the template strand appropriately complexed with the primer. DNA polymerase will be added to complete the process of repair replication. Two molecules of the original duplex should result. The whole cycle could be repeated, there being added every time a fresh dose of the enzyme.’ The above paragraph was written in 1971 [ l ] and describes the principle of what was to be developed into the polymerase chain reaction (PCR). This now well-known procedure generates millions of copies of a specific genomic fragment by exponential amplification without cloning in host cells. However, it was not until 1985 that Saiki et al. [2] actually reported the enzymatic in-vitro amplification of 8-globin genomic sequences and their subsequent analysis. The Abbreviations : AS0 = allele-specific oligonucleotide, DNA = deoxyribonucleic acid, HIV = human immunodeficiency virus, mRNA = messenger ribonucleic acid, PCR = polymerase chain reaction. RFLP = restriction fragment length polymorphism.
unusual circumstances of this rediscovery of the method have been described in a scientific short story [3]. The specificity of PCR is achieved by the use of two synthetic oligonucleotides which hybridize to opposite strands of the deoxyribonucleic acid (DNA). These ‘ primers ’ flank the target sequence with their 3’ ends facing inward. Typically 30 cycles of denaturation of the double-stranded DNA, annealing of the oligonucleotides and extension of these ‘ primers ’ by the DNA polymerase are sufficient to visualize the PCR product in a simple ethidium bromide-stained agarose gel. Use of the thermolabile Klenow enzyme for the extension step required the addition of fresh enzyme after each denaturation step. The introduction of a thermostable polymerase from Thermus aquaticus (Taq DNA polymerase) [4] greatly simplified the experimental procedure [ 51, permitted automation, and led to the rapid development of a wide range of applications, both in biology and in medicine [61. The markedly higher replication error rate of Taqpolymerase, compared to Klenow enzyme [71, does not normally affect the reliability of PCR-based diagnostics [8]. Medical applications therefore ac391
392
J.REISS
count for the majority of published reports [9]. This review is intended to summarize briefly the current and emerging clinical potential of PCR.
Applications In the early days of PCR, reaction tubes were transferred manually between heating blocks maintained at different temperatures. Research workers rapidly became tired of this monotonous procedure and used electronically controlled robot arms for automation. Subsequently, dozens of so-called ‘ thermocyclers ’ have been developed, which temporally change the temperature of the reaction tubes in a 6xed position. Heat-stable polymerases have also been isolated from several thermophilic prokaryotes. In principle, the test for the presence or absence of a specified DNA molecule (called the template or target) should be the most straightforward application of PCR. Genomic sequences can be amplified from even a single cell [lo]. However, due to the extreme sensitivity of the method, this process is highly vulnerable to cross-contamination (which leads to false-positive results). On the other hand, a number of ingredients and appropriate reaction conditions are required for successful amplification. One missing component or incorrect parameter will be sufficient to cause reaction failure (leading to false-negative data). The parallel processing of positive and negative controls, distributing aliquots of the premixed reaction components to the individual test samples, is absolutely obligatory [ l l ] . Further recommendations deal with separated laboratory compartments for pre- and post-amplification procedures or UV/nuclease treatment of the samples before amplification, and specialized instruments [121. The problem of false-positive and false-negative signals is nowhere more apparent than in the widespread testing for human immunodeficiency virus (HIV) sequences [13]. Other applications for various forms of infectious disease, including Hepatitis B, Papillomavirus, Legionella pneurnophila and Trypanosoma cruzi have been summarized elsewhere ~41. The + / - principle of detection of specific sequences is also used to identify deletions that cause inherited disease, e.g. in the Duchenne or Becker muscular dystrophies. Since here the template copy numbers in positive samples are virtually unlimited, the characterization of deletions in hemizygous
patients is reasonably well established [15, 161. The effect of maternal cell contamination on prenatal diagnosis has been studied, and does not appear to interfere with diagnostic reliability, if the contamination rate is not unreasonably high and the experiments are performed with the necessary care [15, 171. However, carrier detection by gene dosage raises the problem of quantitative PCR. Different strategies for such applications have been reported, but in general they do not seem to be feasible for a routine application [18-20]. Other criteria must be applied if PCR is not used as the discriminating step per se, but merely to enrich templates prior to their analysis. Since discrimination between two alleles is sufficient for the analysis of binary patterns (as in genotyping), the advantages of PCR became most apparent in direct and indirect DNA diagnosis. For example, investigation of restriction fragment length polymorphisms (RFLPs) formerly required Southern blotting and hybridization. This technique is laborious and time-consuming. Furthermore, it usually implies the use of radioactive substrates, and the results are not obtained until after 1 week. Williams et a!. [21], in contrast, described a same-day PCR-based diagnosis for cystic fibrosis. The development of PCR-based diagnosis for several common inherited diseases, e.g. sickle cell anaemia, alpha- 1-antitrypsin deficiency and the haemophilias, has been described elsewhere ~91. Cystic fibrosis provides another excellent example of the progress of direct DNA analysis by PCR. The importance of identification of the disease-causing mutations for differential diagnosis cannot be overestimated. The rapid accumulation of international data on the type and frequency of mutations in the ‘cystic fibrosis transmembrane conductance regulator gene’ following its recent cloning [22] would have been impossible without PCR. In the majority of these studies, a previously identified mutation was tested for, but PCR has recently been supplemented by other powerful methods for identification of unknown mutations. This includes direct sequencing of single-stranded (asymmetrical) PCR products [2 31, chemical mismatch cleavage [24-261, and SSCP analysis [27]. The identification of unknown mutations might be further simplified by substituting the compact messenger ribonucleic acid (mRNA) for the complex genomic DNA of eukaryotes in the analyses. This compact target without introns contains most of the
PCR IN DIAGNOSTICS AND RESEARCH
essential gene regions, and can be reverse-transcribed into complementary DNA. The complementary DNA produced serves as a template in a ‘Northern PCR’. The need for expressing tissue in this approach has been eliminated by the detection of ectopic transcripts in non-expressing tissues [28. 291. The general suitability of readily obtainable lymphocytes from peripheral blood as a source for these rare transcripts has been demonstrated by the amplification of spermatogenesis-specific transcripts in adult females [30]. The diagnostic potential of the procedure has been illustrated by studies on haemophilia A [3 1, 321 and the muscular dystrophies [33]. The potential of PCR in cancer diagnostics has been demonstrated by the detection of tumourspecific translocations using either mRNA [34] or genomic DNA [35] as a template. The high suitability of PCR for monitoring patients after treatment has recently been highlighted by the identification of p53 gene mutations in bladder cancers using urine samples [36].
Technical variations The investigation of DNA polymorphisms in both Southern blot and PCR procedures usually requires either restriction enzyme incubation or. if no restriction site is affected, hybridization with allelespecific oligonucleotides (ASOs). The latter might be circumvented by the artificial creation of a RFLP system. One of the PCR primers must therefore carry a mismatched base, which does not interfere with successful amplification and which creates a restriction site in combination with one of the two template alleles [37]. The analysis can be further simplified by using ASOs for the PCR itself. Appropriate primers only allow amplification of one allele or the other, and thus combine allele discrimination and the enrichment step. This principle has been termed allele-specific PCR [38], or the amplification refractory mutation system [39]. The major limitation of ‘standard’ PCR is the requirement for sequence data on both sides of the target region for the synthesis of oligonucleotide primers. With the description of the so-called inverse PCR, this problem appears to have been eliminated [40.41]. This ‘chromosome crawling’ affords enzymatic digestion and religation of genomic DNA prior to amplification. The absence of many application reports, the comments of Fors et al. [42], and observations in my own laboratory indicate that
393
this reaction, at least in the case of the complex eukaryotic and particularly the human genome, is not very practicable. More promising in this respect is the so-called ‘ anchored ’ PCR, which implies one sequence-specific primer and homopolymer annealing to the ‘tailed’ end of the target fragment to which appropriate nucleotides have been added [43].
Future prospects As more and more genes responsible for clinical phenotypes are cloned, sequenced, and their mutational spectra studied, PCR techniques will in turn contribute increasingly to diagnosis and therapeutic monitoring. However, only a small fraction of the human genes involved in hereditary disease have been fully characterized. For those cases in which the search for the disease gene has not yet been completed, the association of high-risk RFLP haplotypes might be exploited in the mean-time. Although successful amplification with as little as a single cell as starting material has been demonstrated, single-copy PCR is largely dependent on the presence of competing DNA molecules. Lo et al. [44], in a tentative study, reported the detection of sequences specific for the Y-chromosome in peripheral blood of pregnant women with a male foetus. Prenatal sex typing by this procedure is at least today beyond the borderline of reliability in PCR experiments. This problem might be circumvented by preimplantation diagnosis, as described by Handyside et al. [45]. However, such novel developments might be judged differently according to national law and ethical background. Forensic studies have surprised us with steadily developing extensions of the potential applications of PCR e.g.. [46, 471. There are several reasons why limitations should be considered, too. Compared to alternative methods PCR is an easy technique, but this does not mean that there are no failures. Use of PCR-derived data for medical purposes must be carried out by experienced individuals who are aware of the drawbacks of the procedure. The trend towards analysis of single-copy templates calls for reconsideration of the effects of incorporation errors. Krawczak et al. [8] estimated the error rate of singlecopy PCR to be of the order of 1 %. Furthermore, the increased possibilities of examining spurious material suggest a corresponding rise in opportunities for misuse. In the future, involuntary probands might not be confined, as now, to criminal cases. Laws will
394
J.REISS
be implemented, hopefully, to restrict the potential negative consequences of PCR applications, and they be not be misguided' History has taught us to be careful with chain reactions.
References 1 Kleppe K. Ohtsuka E. Kleppe R. Molineux I. Khorana HG. Studies on polynucleotides XCVI. Repair replication of short synthetic DNAs as catalyzed by DNA polymerases. I Mol Biol 1 9 7 1 : 56: 341-61. 2 Saiki RK. Scharf S. Faloona F et al. Enzymatic amplification of p-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1 9 8 5 : 230: 1350-54. 3 Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am 1 9 9 0 : April: 36-43. 4 Chien A, Edgar DB, Trela JM. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 1 9 7 6 : 1 2 7 : 1550-57. 5 Kogan SC. Doherty M, Gitschier J. An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. N Engl] Med 1 9 8 7 ; 3 1 7 : 985-90. 6 Vosberg HP. The polymerase chain reaction: an improved method for the analysis of nucleic acids. Hum Genet 1 9 8 9 : 8 3 : 1-15. 7 Tindall KR. Kunkel TA. Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 1988 : 2 7 : 6008-1 3. 8 Krawczak M. Reiss J. Schmidtke J. Rosler U. Polymerase chain reaction : replication errors and reliability of gene diagnosis. Nucleic Acids Res 1 9 8 9 : 1 7 : 2197-201. 9 Reiss J, Cooper DN. Application of the polymerase chain reaction to the diagnosis of human genetic disease. Hum Genet 1 9 9 0 ; 8 5 : 1-8. 1 0 Li H. Gyllenstein UB. Cui X. Saiki RK. Erlich HA, Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 1 9 8 8 : 335: 414-7. 11 Reiss J. Neufeld U. Wieland K. Zoll B. Diagnosis of haemophilia B using the polymerase chain reaction. Blut 1 9 9 0 : 60: 31-6. 1 2 Orrego C. Organizing a laboratory for PCR work. In: Innis MA, Gelfand DH. Sninsky JJ, White TJ, eds. PCR Protocols: A Guide to Methods and Applications. San Diego : Academic Press Inc., 1990: 447-54. 13 Kellogg DE. Kwok S. Detection of human immunodeficiency virus. In: Innis MA, Gelfand DH. Sninsky JJ. White Tj. eds. PCR Protocols: A Guide to Methods and Applications. San Diego: Academic Press Inc., 1990; 337-47. 1 4 Eisenstein BI. The polymerase chain reaction - a new method of using molecular genetics for medical diagnosis. N Engl Med 1990: 322: 178-83. 1 5 Chamberlain JS. Gibbs RA. Ranier JE, Nguyen PN, Caskey CT. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res 1988: 2 3 : 11141-56. 1 6 Beggs AH, Koenig M. Boyce FM. Kunkel LM. Detection of 9 8 % of DMD/BMD gene deletions by polymerase chain reaction. Hum Genet 1 9 9 0 ; 8 6 : 45-8. 1 7 Hentemann M. Reiss, J. Wagner M. Cooper DN. Rapid detection of deletions in the Duchenne muscular dystrophy gene by amplification of deletion-prone exon sequences. Hum Genet 1 9 9 0 : 8 4 : 228-32.
1 8 Chelly J, Kaplan JC, Maire P. Gautron S. Kahn A. Transcription of the dystrophin gene in human muscle and non-muscle tissues. Nature 1988: 333: 858-60. 1 9 Gilliland G, Perrin S, Blanchard K, Bunn HF. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci USA 1 9 9 0 : 8 7 : 2725-9. 20 Gaudette MF. Crain WR. A simple method for quantifying specific mRNAs in small numbers of early mouse embryos. Nucleic Acids Res 1991 : 1 9 : 1879-84. 21 Williams C. Williams R, Coutelle C. Loeffler F. Smith J. Ivinson A. Same-day, first-trimester antenatal diagnosis for cystic fibrosis by gene amplification. h n c e t 1 9 8 8 : ii: 102-3. 22 Riordan JR. Rommens JM. Kerem BS el al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1 9 8 9 : 245: 1066-73. 23 Cyllensten UB, Erlich HA. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci USA 1988: 8 5 : 7652-6. 2 4 Cotton RGH. Rodrigues NR, Campbell RD. Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Nut1 Acad Sci USA 1 9 8 8 ; 8 5 : 4397402. 25 Montandon AJ. Green PM. Gianelli F. Bentley DR. Direct detection ofpoint mutations by mismatch analysis: application to haemophilia B. Nucleic Acids Res 1989: 1 7 : 3347-58. 26 Grompe M. Muzny DM. Caskey CT. Scanning detection of mutations in human ornithine transcarbamoylase by chemical mismatch cleavage. Proc Natl Acad Sci USA 1 9 8 9 : 8 6 : 5 8 88-9 2. 2 7 Orita M, Suzuki Y . Sekiya T, Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 1989: 5: 874-9. 28 Chelly J. Concordet JP. Kaplan JC,Kahn A. Illegitimate transcription : transcription of any gene in any cell type. Proc Natl Acad Sci USA 1989: 8 6 : 2617-21. 29 Sarkar G. Sommer SS. Excess to a messenger RNA sequence or its protein product is not limited by tissue or species specificity. Science 1 9 8 9 ; 244: 3 3 1 4 . 3 0 Slomski R. Schlosser M. Chlebowska H. Reiss J. Engel W. Detection of human spermatid-specific transcripts in the lymphocytes of males and females. Hum Genet 1991 : 8 7 : 307-1 0. 31 Berg LP, Wieland K. Miller DS et a!. Detection of a novel point mutation causing haemophilia A by PCR/direct sequencing of ectopically-transcribed Factor VITI mRNA. Hum Genet 1990 ; 8 5 : 655-8. 32 Naylor JA. Green PM, Montandon AJ. Rizza CR. Gianella F. Detection of three novel mutations in two haemophilia A patients by rapid screening of whole essential region of factor VlII gene. Lancet 1991 : 337: 635-9. 33 Schloesser M. Slomski R. Wagner M et a/. Characterization of pathological dystrophin transcripts from the lymphocytes of a muscular dystrophy carrier. Mol Biol Med 1 9 9 0 : 7 : 519-23. 3 4 Kawasaki ES. Clark SS. Coyne MY et al. Diagnoses of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro. Proc Natl Acad Sci USA 1988: 8 5 : 5698-702. 35 Lee MS, Chang KS, Cabanillas F. Freireich El,Trujillo JM. Stass SA. Detection of minimal residual cells carrying the t (14: 18) by DNA sequence amplification. Science 1 9 8 7 : 237: 175-8.
PCR IN DIAGNOSTICS A N D RESEARCH
36 Sidranski D. Eschenbach A, Tsai YC et al. Identification of p53 gene mutations in bladder cancers and urine samples. Science 1 9 9 1 : 252: 706-9. 3 7 Haliassos A, Chomel J. Tesson A el al. Modification of enzymatically amplified DNA for the detection of point mutations. Nucleic Acids Res 1 9 8 9 : 1 7 : 3606. 38 Wu DY. Ugolozzi L. Pal BK. Wallace RB. Allele-specific enzymatic amplification ofg-globin genomic DNA for diagnosis of sickle cell anemia. Proc Nut1 Acad Sci U S A 1989: 86: 2757-60. 39 Newton CR, Graham A, Heptinstall LE et a/. Analysis of any point mutation in DNA. The amplification refractory mutation system. Nucleic Acids Res 1989: 17: 2503-16. 40 Ochman H. Gerber AS, Hart1 DL. Genetic applications of a n inverse polymerase chain reaction. Grnetics 1 9 8 8 : 120: 621-3. 41 Triglia T. Peterson MG. Kemp DJ. A procedure for in-vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res 1 9 8 8 : 16: 81 86. 4 2 Fors L. Saavedra RA. Hood L. Cloning of the shark Po promotor using a genomic walking technique based on the
43
44
45
46
47
395
polymerase chain reaction. Nucleic Acids Res 1 9 9 0 : 18: 2793-9. Loh EY. Elliot JF. Cwirla S et al. Polynierase chain reaction with single-sided specificity : analysis of T-cell receptor delta chain. Science 1 9 8 9 ; 243: 217-20. Lo YMD. Pate1 P. Wainscoat JS, Sampietro M. Gillmer MDG, Fleming KA. Prenatal sex determination by DNA amplification from maternal peripheral blood. Lancet 1989: ii: 1363-5. Handyside AH, Penketh RJA. Winston RML et a/. Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet 1 9 8 9 ; ii: 347-9. Jeffreys AJ, Wilson V, Neumann R, Keyte 1. Amplification of human minisatellites by the polymerase chain reaction : towards DNA fingerprinting of single cells. Nucleic Acids Res 1 9 8 8 : 16: 10953-71. Higuchi R, Beroldingen CH. Sensabaugh GF, Erlich HA. DNA typing from single hairs. Nature 1 9 8 8 : 332: 543-6.
Received 11 June 1991, accepted 5 ]uly 1991. Correspondence: J. Reiss. MD PhD. Institut fur Humangenetik der Universitat, D-3400 Gottingen. Germany.
Review article The polymerase chain reaction and its potential role in clinical diagnostics and research J. REISS From the Institut f u r Hurnangenetik der Universitat. Giittingen. Germany
Abstract. Reiss J (Institut fur Humangenetik der Universitat, Gottingen. Germany). The polymerase Ehain reaction and its potential role in clinical diagnostics and research. journal of Internal Medicine 1991: 230: 391-395.
In-vitro amplification of deoxyribonucleic acid molecules by means of the polymerase chain reaction (PCR) must be regarded as the most important advance in the life sciences to take place during the last decade. Originally applied to the identification of mutations in well-known and fully sequenced human genes, its applications have now been extended to a wide variety of biological and medical disciplines, accompanied by significant technical improvements and sophisticated variations of the basic principle. Specialized molecular genetics laboratories were the first to employ this new method, and they still are in the process of extending its potential. Due to its unique properties, applications of PCR quickly spread to other areas of research, and numerous clinical studies have already employed PCR. The field is currently still expanding rapidly. Keywords : clinical research, diagnostics, DNA, polymerase chain reaction.
Introduction ‘The D N A duplex would be denatured to form single strands. This denaturation step would be carried out in the presence of a sujjiciently large excess of the two appropriate primers. Upon cooling, one would hope to obtain two structures, each containing the full length of the template strand appropriately complexed with the primer. DNA polymerase will be added to complete the process of repair replication. Two molecules of the original duplex should result. The whole cycle could be repeated, there being added every time a fresh dose of the enzyme.’ The above paragraph was written in 1971 [ l ] and describes the principle of what was to be developed into the polymerase chain reaction (PCR). This now well-known procedure generates millions of copies of a specific genomic fragment by exponential amplification without cloning in host cells. However, it was not until 1985 that Saiki et al. [2] actually reported the enzymatic in-vitro amplification of 8-globin genomic sequences and their subsequent analysis. The Abbreviations : AS0 = allele-specific oligonucleotide, DNA = deoxyribonucleic acid, HIV = human immunodeficiency virus, mRNA = messenger ribonucleic acid, PCR = polymerase chain reaction. RFLP = restriction fragment length polymorphism.
unusual circumstances of this rediscovery of the method have been described in a scientific short story [3]. The specificity of PCR is achieved by the use of two synthetic oligonucleotides which hybridize to opposite strands of the deoxyribonucleic acid (DNA). These ‘ primers ’ flank the target sequence with their 3’ ends facing inward. Typically 30 cycles of denaturation of the double-stranded DNA, annealing of the oligonucleotides and extension of these ‘ primers ’ by the DNA polymerase are sufficient to visualize the PCR product in a simple ethidium bromide-stained agarose gel. Use of the thermolabile Klenow enzyme for the extension step required the addition of fresh enzyme after each denaturation step. The introduction of a thermostable polymerase from Thermus aquaticus (Taq DNA polymerase) [4] greatly simplified the experimental procedure [ 51, permitted automation, and led to the rapid development of a wide range of applications, both in biology and in medicine [61. The markedly higher replication error rate of Taqpolymerase, compared to Klenow enzyme [71, does not normally affect the reliability of PCR-based diagnostics [8]. Medical applications therefore ac391
392
J.REISS
count for the majority of published reports [9]. This review is intended to summarize briefly the current and emerging clinical potential of PCR.
Applications In the early days of PCR, reaction tubes were transferred manually between heating blocks maintained at different temperatures. Research workers rapidly became tired of this monotonous procedure and used electronically controlled robot arms for automation. Subsequently, dozens of so-called ‘ thermocyclers ’ have been developed, which temporally change the temperature of the reaction tubes in a 6xed position. Heat-stable polymerases have also been isolated from several thermophilic prokaryotes. In principle, the test for the presence or absence of a specified DNA molecule (called the template or target) should be the most straightforward application of PCR. Genomic sequences can be amplified from even a single cell [lo]. However, due to the extreme sensitivity of the method, this process is highly vulnerable to cross-contamination (which leads to false-positive results). On the other hand, a number of ingredients and appropriate reaction conditions are required for successful amplification. One missing component or incorrect parameter will be sufficient to cause reaction failure (leading to false-negative data). The parallel processing of positive and negative controls, distributing aliquots of the premixed reaction components to the individual test samples, is absolutely obligatory [ l l ] . Further recommendations deal with separated laboratory compartments for pre- and post-amplification procedures or UV/nuclease treatment of the samples before amplification, and specialized instruments [121. The problem of false-positive and false-negative signals is nowhere more apparent than in the widespread testing for human immunodeficiency virus (HIV) sequences [13]. Other applications for various forms of infectious disease, including Hepatitis B, Papillomavirus, Legionella pneurnophila and Trypanosoma cruzi have been summarized elsewhere ~41. The + / - principle of detection of specific sequences is also used to identify deletions that cause inherited disease, e.g. in the Duchenne or Becker muscular dystrophies. Since here the template copy numbers in positive samples are virtually unlimited, the characterization of deletions in hemizygous
patients is reasonably well established [15, 161. The effect of maternal cell contamination on prenatal diagnosis has been studied, and does not appear to interfere with diagnostic reliability, if the contamination rate is not unreasonably high and the experiments are performed with the necessary care [15, 171. However, carrier detection by gene dosage raises the problem of quantitative PCR. Different strategies for such applications have been reported, but in general they do not seem to be feasible for a routine application [18-20]. Other criteria must be applied if PCR is not used as the discriminating step per se, but merely to enrich templates prior to their analysis. Since discrimination between two alleles is sufficient for the analysis of binary patterns (as in genotyping), the advantages of PCR became most apparent in direct and indirect DNA diagnosis. For example, investigation of restriction fragment length polymorphisms (RFLPs) formerly required Southern blotting and hybridization. This technique is laborious and time-consuming. Furthermore, it usually implies the use of radioactive substrates, and the results are not obtained until after 1 week. Williams et a!. [21], in contrast, described a same-day PCR-based diagnosis for cystic fibrosis. The development of PCR-based diagnosis for several common inherited diseases, e.g. sickle cell anaemia, alpha- 1-antitrypsin deficiency and the haemophilias, has been described elsewhere ~91. Cystic fibrosis provides another excellent example of the progress of direct DNA analysis by PCR. The importance of identification of the disease-causing mutations for differential diagnosis cannot be overestimated. The rapid accumulation of international data on the type and frequency of mutations in the ‘cystic fibrosis transmembrane conductance regulator gene’ following its recent cloning [22] would have been impossible without PCR. In the majority of these studies, a previously identified mutation was tested for, but PCR has recently been supplemented by other powerful methods for identification of unknown mutations. This includes direct sequencing of single-stranded (asymmetrical) PCR products [2 31, chemical mismatch cleavage [24-261, and SSCP analysis [27]. The identification of unknown mutations might be further simplified by substituting the compact messenger ribonucleic acid (mRNA) for the complex genomic DNA of eukaryotes in the analyses. This compact target without introns contains most of the
PCR IN DIAGNOSTICS AND RESEARCH
essential gene regions, and can be reverse-transcribed into complementary DNA. The complementary DNA produced serves as a template in a ‘Northern PCR’. The need for expressing tissue in this approach has been eliminated by the detection of ectopic transcripts in non-expressing tissues [28. 291. The general suitability of readily obtainable lymphocytes from peripheral blood as a source for these rare transcripts has been demonstrated by the amplification of spermatogenesis-specific transcripts in adult females [30]. The diagnostic potential of the procedure has been illustrated by studies on haemophilia A [3 1, 321 and the muscular dystrophies [33]. The potential of PCR in cancer diagnostics has been demonstrated by the detection of tumourspecific translocations using either mRNA [34] or genomic DNA [35] as a template. The high suitability of PCR for monitoring patients after treatment has recently been highlighted by the identification of p53 gene mutations in bladder cancers using urine samples [36].
Technical variations The investigation of DNA polymorphisms in both Southern blot and PCR procedures usually requires either restriction enzyme incubation or. if no restriction site is affected, hybridization with allelespecific oligonucleotides (ASOs). The latter might be circumvented by the artificial creation of a RFLP system. One of the PCR primers must therefore carry a mismatched base, which does not interfere with successful amplification and which creates a restriction site in combination with one of the two template alleles [37]. The analysis can be further simplified by using ASOs for the PCR itself. Appropriate primers only allow amplification of one allele or the other, and thus combine allele discrimination and the enrichment step. This principle has been termed allele-specific PCR [38], or the amplification refractory mutation system [39]. The major limitation of ‘standard’ PCR is the requirement for sequence data on both sides of the target region for the synthesis of oligonucleotide primers. With the description of the so-called inverse PCR, this problem appears to have been eliminated [40.41]. This ‘chromosome crawling’ affords enzymatic digestion and religation of genomic DNA prior to amplification. The absence of many application reports, the comments of Fors et al. [42], and observations in my own laboratory indicate that
393
this reaction, at least in the case of the complex eukaryotic and particularly the human genome, is not very practicable. More promising in this respect is the so-called ‘ anchored ’ PCR, which implies one sequence-specific primer and homopolymer annealing to the ‘tailed’ end of the target fragment to which appropriate nucleotides have been added [43].
Future prospects As more and more genes responsible for clinical phenotypes are cloned, sequenced, and their mutational spectra studied, PCR techniques will in turn contribute increasingly to diagnosis and therapeutic monitoring. However, only a small fraction of the human genes involved in hereditary disease have been fully characterized. For those cases in which the search for the disease gene has not yet been completed, the association of high-risk RFLP haplotypes might be exploited in the mean-time. Although successful amplification with as little as a single cell as starting material has been demonstrated, single-copy PCR is largely dependent on the presence of competing DNA molecules. Lo et al. [44], in a tentative study, reported the detection of sequences specific for the Y-chromosome in peripheral blood of pregnant women with a male foetus. Prenatal sex typing by this procedure is at least today beyond the borderline of reliability in PCR experiments. This problem might be circumvented by preimplantation diagnosis, as described by Handyside et al. [45]. However, such novel developments might be judged differently according to national law and ethical background. Forensic studies have surprised us with steadily developing extensions of the potential applications of PCR e.g.. [46, 471. There are several reasons why limitations should be considered, too. Compared to alternative methods PCR is an easy technique, but this does not mean that there are no failures. Use of PCR-derived data for medical purposes must be carried out by experienced individuals who are aware of the drawbacks of the procedure. The trend towards analysis of single-copy templates calls for reconsideration of the effects of incorporation errors. Krawczak et al. [8] estimated the error rate of singlecopy PCR to be of the order of 1 %. Furthermore, the increased possibilities of examining spurious material suggest a corresponding rise in opportunities for misuse. In the future, involuntary probands might not be confined, as now, to criminal cases. Laws will
394
J.REISS
be implemented, hopefully, to restrict the potential negative consequences of PCR applications, and they be not be misguided' History has taught us to be careful with chain reactions.
References 1 Kleppe K. Ohtsuka E. Kleppe R. Molineux I. Khorana HG. Studies on polynucleotides XCVI. Repair replication of short synthetic DNAs as catalyzed by DNA polymerases. I Mol Biol 1 9 7 1 : 56: 341-61. 2 Saiki RK. Scharf S. Faloona F et al. Enzymatic amplification of p-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1 9 8 5 : 230: 1350-54. 3 Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am 1 9 9 0 : April: 36-43. 4 Chien A, Edgar DB, Trela JM. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 1 9 7 6 : 1 2 7 : 1550-57. 5 Kogan SC. Doherty M, Gitschier J. An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. N Engl] Med 1 9 8 7 ; 3 1 7 : 985-90. 6 Vosberg HP. The polymerase chain reaction: an improved method for the analysis of nucleic acids. Hum Genet 1 9 8 9 : 8 3 : 1-15. 7 Tindall KR. Kunkel TA. Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 1988 : 2 7 : 6008-1 3. 8 Krawczak M. Reiss J. Schmidtke J. Rosler U. Polymerase chain reaction : replication errors and reliability of gene diagnosis. Nucleic Acids Res 1 9 8 9 : 1 7 : 2197-201. 9 Reiss J, Cooper DN. Application of the polymerase chain reaction to the diagnosis of human genetic disease. Hum Genet 1 9 9 0 ; 8 5 : 1-8. 1 0 Li H. Gyllenstein UB. Cui X. Saiki RK. Erlich HA, Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 1 9 8 8 : 335: 414-7. 11 Reiss J. Neufeld U. Wieland K. Zoll B. Diagnosis of haemophilia B using the polymerase chain reaction. Blut 1 9 9 0 : 60: 31-6. 1 2 Orrego C. Organizing a laboratory for PCR work. In: Innis MA, Gelfand DH. Sninsky JJ, White TJ, eds. PCR Protocols: A Guide to Methods and Applications. San Diego : Academic Press Inc., 1990: 447-54. 13 Kellogg DE. Kwok S. Detection of human immunodeficiency virus. In: Innis MA, Gelfand DH. Sninsky JJ. White Tj. eds. PCR Protocols: A Guide to Methods and Applications. San Diego: Academic Press Inc., 1990; 337-47. 1 4 Eisenstein BI. The polymerase chain reaction - a new method of using molecular genetics for medical diagnosis. N Engl Med 1990: 322: 178-83. 1 5 Chamberlain JS. Gibbs RA. Ranier JE, Nguyen PN, Caskey CT. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res 1988: 2 3 : 11141-56. 1 6 Beggs AH, Koenig M. Boyce FM. Kunkel LM. Detection of 9 8 % of DMD/BMD gene deletions by polymerase chain reaction. Hum Genet 1 9 9 0 ; 8 6 : 45-8. 1 7 Hentemann M. Reiss, J. Wagner M. Cooper DN. Rapid detection of deletions in the Duchenne muscular dystrophy gene by amplification of deletion-prone exon sequences. Hum Genet 1 9 9 0 : 8 4 : 228-32.
1 8 Chelly J, Kaplan JC, Maire P. Gautron S. Kahn A. Transcription of the dystrophin gene in human muscle and non-muscle tissues. Nature 1988: 333: 858-60. 1 9 Gilliland G, Perrin S, Blanchard K, Bunn HF. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci USA 1 9 9 0 : 8 7 : 2725-9. 20 Gaudette MF. Crain WR. A simple method for quantifying specific mRNAs in small numbers of early mouse embryos. Nucleic Acids Res 1991 : 1 9 : 1879-84. 21 Williams C. Williams R, Coutelle C. Loeffler F. Smith J. Ivinson A. Same-day, first-trimester antenatal diagnosis for cystic fibrosis by gene amplification. h n c e t 1 9 8 8 : ii: 102-3. 22 Riordan JR. Rommens JM. Kerem BS el al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1 9 8 9 : 245: 1066-73. 23 Cyllensten UB, Erlich HA. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci USA 1988: 8 5 : 7652-6. 2 4 Cotton RGH. Rodrigues NR, Campbell RD. Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Nut1 Acad Sci USA 1 9 8 8 ; 8 5 : 4397402. 25 Montandon AJ. Green PM. Gianelli F. Bentley DR. Direct detection ofpoint mutations by mismatch analysis: application to haemophilia B. Nucleic Acids Res 1989: 1 7 : 3347-58. 26 Grompe M. Muzny DM. Caskey CT. Scanning detection of mutations in human ornithine transcarbamoylase by chemical mismatch cleavage. Proc Natl Acad Sci USA 1 9 8 9 : 8 6 : 5 8 88-9 2. 2 7 Orita M, Suzuki Y . Sekiya T, Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 1989: 5: 874-9. 28 Chelly J. Concordet JP. Kaplan JC,Kahn A. Illegitimate transcription : transcription of any gene in any cell type. Proc Natl Acad Sci USA 1989: 8 6 : 2617-21. 29 Sarkar G. Sommer SS. Excess to a messenger RNA sequence or its protein product is not limited by tissue or species specificity. Science 1 9 8 9 ; 244: 3 3 1 4 . 3 0 Slomski R. Schlosser M. Chlebowska H. Reiss J. Engel W. Detection of human spermatid-specific transcripts in the lymphocytes of males and females. Hum Genet 1991 : 8 7 : 307-1 0. 31 Berg LP, Wieland K. Miller DS et a!. Detection of a novel point mutation causing haemophilia A by PCR/direct sequencing of ectopically-transcribed Factor VITI mRNA. Hum Genet 1990 ; 8 5 : 655-8. 32 Naylor JA. Green PM, Montandon AJ. Rizza CR. Gianella F. Detection of three novel mutations in two haemophilia A patients by rapid screening of whole essential region of factor VlII gene. Lancet 1991 : 337: 635-9. 33 Schloesser M. Slomski R. Wagner M et a/. Characterization of pathological dystrophin transcripts from the lymphocytes of a muscular dystrophy carrier. Mol Biol Med 1 9 9 0 : 7 : 519-23. 3 4 Kawasaki ES. Clark SS. Coyne MY et al. Diagnoses of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro. Proc Natl Acad Sci USA 1988: 8 5 : 5698-702. 35 Lee MS, Chang KS, Cabanillas F. Freireich El,Trujillo JM. Stass SA. Detection of minimal residual cells carrying the t (14: 18) by DNA sequence amplification. Science 1 9 8 7 : 237: 175-8.
PCR IN DIAGNOSTICS A N D RESEARCH
36 Sidranski D. Eschenbach A, Tsai YC et al. Identification of p53 gene mutations in bladder cancers and urine samples. Science 1 9 9 1 : 252: 706-9. 3 7 Haliassos A, Chomel J. Tesson A el al. Modification of enzymatically amplified DNA for the detection of point mutations. Nucleic Acids Res 1 9 8 9 : 1 7 : 3606. 38 Wu DY. Ugolozzi L. Pal BK. Wallace RB. Allele-specific enzymatic amplification ofg-globin genomic DNA for diagnosis of sickle cell anemia. Proc Nut1 Acad Sci U S A 1989: 86: 2757-60. 39 Newton CR, Graham A, Heptinstall LE et a/. Analysis of any point mutation in DNA. The amplification refractory mutation system. Nucleic Acids Res 1989: 17: 2503-16. 40 Ochman H. Gerber AS, Hart1 DL. Genetic applications of a n inverse polymerase chain reaction. Grnetics 1 9 8 8 : 120: 621-3. 41 Triglia T. Peterson MG. Kemp DJ. A procedure for in-vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res 1 9 8 8 : 16: 81 86. 4 2 Fors L. Saavedra RA. Hood L. Cloning of the shark Po promotor using a genomic walking technique based on the
43
44
45
46
47
395
polymerase chain reaction. Nucleic Acids Res 1 9 9 0 : 18: 2793-9. Loh EY. Elliot JF. Cwirla S et al. Polynierase chain reaction with single-sided specificity : analysis of T-cell receptor delta chain. Science 1 9 8 9 ; 243: 217-20. Lo YMD. Pate1 P. Wainscoat JS, Sampietro M. Gillmer MDG, Fleming KA. Prenatal sex determination by DNA amplification from maternal peripheral blood. Lancet 1989: ii: 1363-5. Handyside AH, Penketh RJA. Winston RML et a/. Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet 1 9 8 9 ; ii: 347-9. Jeffreys AJ, Wilson V, Neumann R, Keyte 1. Amplification of human minisatellites by the polymerase chain reaction : towards DNA fingerprinting of single cells. Nucleic Acids Res 1 9 8 8 : 16: 10953-71. Higuchi R, Beroldingen CH. Sensabaugh GF, Erlich HA. DNA typing from single hairs. Nature 1 9 8 8 : 332: 543-6.
Received 11 June 1991, accepted 5 ]uly 1991. Correspondence: J. Reiss. MD PhD. Institut fur Humangenetik der Universitat, D-3400 Gottingen. Germany.
