PATHOLOGY PRACTICE
Fetal Pediatr Pathol Downloaded from informahealthcare.com by York University Libraries on 12/30/14 For personal use only.
POLYMERASE CHAIN REACTION
Thomas A. Seemayer, MD 0 The Nicolas W. Matossian Molecular Pathology Laboratory, The Montreal Children’s Hospital, McGill University Faculty of Medicine, Montreal, Quebec H3H 1P3, Canada
INTRODUCTION Over the past several decades, molecular biology has assumed a position in the forefront of medical science. Nowadays many of the advances in medicine have a decided molecular slant. These contributions have been made possible by fundamental technological and conceptual breakthroughs, which have facilitated the dissection of molecular events central to both health and disease. Among many of these advances, several landmarks come to mind. Not surprisingly, these advances are linked, since one discovery often facilitated those that followed. I n 1970 restriction endonucleases were identified in bacteria (1). The significance of this discovery (akin to the elucidation of the structure of DNA itself) was recognized immediately, for scientists could thereby utilize minute quantities of these enzymes to cleave DNA with precision, thereby generating a multitude of defined segments. This labor led to the birth of molecular cloning, which became a reality in 1973 (2). It then became possible to insert and ligate, virtually at will, small defined DNA segments into bacterial plasmids or phages to produce copious (in molecular terms) amounts of highly purified DNA. For those working (at that time) with DNA in agarose gels, the report of Southern in 1975 describing a simple method for transferring DNA from agarose to filters surely represented a major contribution (3). This procedure also reduced the DNA requirement for most experiments about tenfold over previous methods. The long-standing technical obstacles to rapid and efficient DNA sequencing were overcome by 1977 (4,5 ) . That year, short segments of DNA were synthesized chemically (6); with this, a new term entered the English language: the “gene machine.’’ In 1978, the Address reprint requests to Thomas A. Seemayer, M.D., Department of Pathology, The Montreal Children’s Hospital, 2300 Tupper Street, Montreal, Quebec H3H 1P3, Canada. Pediatric Pathology, 10.311-31 7, 1990 Copyright 0 1990 by Hemisphere Publishing Corporation
31 1
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312
T. H. SEEMAYER
discovery of DNA-sequence polymorphisms adjacent to the human P-globin structural gene (7) set the stage for the broad application of restriction fragment length polymorphisms (RFLPs) (8) as molecular genetic markers for a vast assortment of diseases. These collective advances made possible the analysis of molecular aberrations central to many human diseases. Early on, the hemoglobinopathies were a prime target of investigation. More recently, diseases such as cystic fibrosis, Duchenne muscular dystrophy, Huntington’s disease, Alzheimer’s disease, schizophrenia, and diverse neoplasms are being understood in a manner never before possible. For the most part these studies require the extraction, purification, electrophoretic separation, and cloning of genomic DNA from blood lymphocytes, chorionic villi, or fragments of fresh or frozen tissue. Clearly the process worked and at the time was considered ideal. Now, however, molecular analysis and cloning call for an amount of source DNA (5-10 mg) and labor (days to weeks) well in excess of that required in light of further technological development.
OVERVIEW OF THE POLYMERASE CHAIN REACTION In 1985, a new and revolutionary procedure, the polymerase chain reaction (PCR), was developed by Mullis and colleagues at the Cetus Corporation (9, 10). As is often the case with discoveries of this magnitude, the principles are relatively simple. The description of this procedure and its applications follow. The PCR, an in-vitro method for the primer-directed enzymatic amplification of specific DNA sequences, mimics normal DNA replication in that each cycle of the reaction results in the doubling of DNA. The process allows for the generation of million of copies of a highly specific (usually short, e.g., 15-400 base pair) DNA segment within hours. The source of DNA to be amplified can be provided by fresh or frozen tissue, blood lymphocytes, chorionic villi, buccal epithelial cells (1 l), hair follicles (12), and even formalinfixed tissue within paraffin blocks. Because DNA has been recovered and cloned from a radiocarbon dated 2,400-year-old Egyptian mummy (1 3), the potential applications of PCR amplification in the study of archival specimens appears limitless. The quantity of DNA required for amplification is infinitesimal: a single hair root with a follicle, a single diploid cell, a single sperm (14), a drop of blood-even a microdissected region from a single chromosome (15) can serve as source material. The ability of the PCR to amplify such minute amounts of target, however, demands that the strictest care be employed to avoid contamination with DNA of an extraneous source. The DNA need not
Fetal Pediatr Pathol Downloaded from informahealthcare.com by York University Libraries on 12/30/14 For personal use only.
POLYMERASE CHAIN REACTION
313
be “in good shape,” that is, purified or of high molecular weight; partiallydegraded DNA will provide a suitable target for amplification provided that there is a sufficient amount of single-stranded DNA to bridge the distance between the 5’ ends of the two primers. Any segment of interest, even a single copy gene buried cryptically amidst the entire human genome, can be amplified. Not only does the procedure work with DNA as source material (either single- or double-stranded), but it also can be applied to RNA. With a minor modification, RNA, when expressed, can be converted to firststrand cDNA with reverse transcriptase and oligoprimers, and amplification can proceed. Because of the magnitude of amplification, radiolabelled probes can be avoided; the amplified segment can be visualized with ethidium bromide (in agarose gels) or nonradiolabelled probes. With a recent refinement of the procedure, the process has been automated. In the end, picograms of source DNA (target and nontarget) are amplified to microgram amounts (target) within several hours. Finally, since amplification is of this level of magnitude, the need for subcloning can be obviated, and the fragments can be sequenced directly. The essence of the PCR resides in the selection of a set (or two sets in the case of “nested”primers) of oligonucleotide primers complementary to a portion of the coding sequence of the segment to be amplified. These primer nucleotide sequences (usually about 20) are synthesized chemically. The oligoprimers flank the segment to be amplified, anneal to their complementary DNA sequences, and direct DNA synthesis in the 5‘ to 3 ‘ direction. Typically, the primers are not complementary to one another; supplied in excess relative to the target DNA template, the formation of primer-template complexes (synthesized in opposite and overlapping directions) is favored over the reassociation of the two DNA strands following the later reduction of temperature. With each cycle, the amount of newly synthesized DNA is doubled, because the extension product of one primer functions as the template for the other. The end of the specific fragment produced is determined by the 5 ’ end of the oligoprimers (which have become incorporated into the product). Although the 5’ end is usually matched to the original template, it may be purposely mismatched, thereby allowing the introduction of other sequences into the product. Thus, insertions, deletions, nucleotide substitutions (in-vitro mutations), regulatory elements, and even restriction site linkers (for later linkage to cloning vectors for sequencing) can be added. With repeated cycles, two DNA products are produced. The “short product,” the region between the 5 ’ ends of the extension primer, is,a discrete, double-stranded DNA molecule produced in great excess. This molecule is of interest because its defined ends correspond to the primers’ sequence. A “long product,” derived from the template molecules in each cycle, is produced in far lesser amounts and is characterized by variable nucleotides at the 3 ’ end (1 6- 19).
314
T. H. SEEMAYER
Fetal Pediatr Pathol Downloaded from informahealthcare.com by York University Libraries on 12/30/14 For personal use only.
THE REACTION The reaction proceeds in three steps (which constitute a cycle), which are repeated over many (20 or more) cycles. The first step, denaturation, brought about by high temperature, separates the two strands of the target DNA molecule. The second, annealing of the extension primers to the DNA template, takes place at a lower temperature (37"-55"C). The third, primer extension with deoxynucleotide triphosphates and DNA polymerase, is performed at a higher temperature (72°C). In the original descriptions (9, 10, 20), the Klenow fragment of E. coli DNA polymerase I was employed at 37 "C. The process was tedious and expensive, because this fragment was thermolabile and inactivated following the heat denaturation step; thus, fresh enzyme had to be added with each cycle. As well, a variety of products was produced, only some of which were specific. In 1987-1988 the process was improved following the substitution of a thermostable DNA polymerase derived from a thermophilic bacterium, Themus aquaticus (Tag) (2 1). This polymerase, usually added only once at the time of the first cycle, is stabile under denaturation conditions and results in a markedly enhanced yield of specific-sized fragments. Single copy sequences have been amplified over 10 millionfold with very high specificity. Moreover, the method has the capability to amplify and detect a target DNA molecule contained in a sample of 100,000 cells. In addition, long specific amplified fragments have been produced (up to 3.2 kilobases) (22, 23). The superiority of this polymerase led directly to the development of a DNA Thermal Cycler (Perkin-Elmer Cetus Instruments), thereby automating the PCR.
POLYMERASE CHAIN REACTION APPLICATIONS The first report of PCR amplification included a clever technique to differentiate between the A and S alleles in the beta chain of human hemoglobin. Following PCR amplification (some 200,000 times) of target DNA, oligomer restriction analysis was performed (10). In this maneuver, the amplified DNA was hybridized to a labelled oligonucleotide probe and restriction enzyme cleavage of the duplex was performed. Because of a designed mismatch within the restriction site in the sickle variant of the beta globin gene and the probe, cleavage did not take place; thus the distinction between the two forms of the hemoglobin molecule was a relatively simple task following electrophoresis and radioautography. The second application established that PCR amplification could be followed by the direct cloning and sequencing of amplified genomic DNA. This advance was brought about with an oligoprimer modification at one of the 5 '
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POLYMERASE CHAIN REACTION
315
ends to produce restriction site linkers. In this report, a 110-base pair fragment of the human P-globin gene and a 242-base pair fragment of the HLAD Q alpha locus were analyzed (24). PCR amplification followed by dot blot analysis with allele-specific oligonucleotide probes (ASO) came next. This is a particularly elegant utilization of the PCR, one with countless applications. These short probes are specific to the extent that a single base pair mismatch negates probe-target recognition (binding). Following the amplification of a 110 bp fragment of the sixth codon of the human 0-globin gene, specific oligoprobes for hemoglobin A, S, and C easily distinguished the hemoglobin content in the samples (20, 25). The amplification was also shown to be effective with crude cell lysates, thereby negating the requirement for DNA purification. As a follow-up, the procedure worked well with DNA extracted from paraffin blocks (26). A similar approach reported about this time employed frozen tissue and oligoprobes to establish the high incidence of c-Ki-ras mutations at codon 12 in colorectal carcinomas (2 7). The first description of Taq polymerase-directed PCR’s came in a study of blood (finger pricking) and chorionic villi biopsies in a setting of hemophilia A focused on carrier detection and prenatal diagnosis (22). When the amplified products were digested, appropriately sized fragments were visualized in the ethidium bromide treated gels. Allele-specific oligoprobes in dot blots were able to distinguish a single base-pair alteration responsible for a BclI polymorphism. Fetal sex was established by amplification of the Y-chromosome 3.4 kb repeats. This study (and one that soon followed) (23) established the superiority of Taq polymerase to the Klenow fragment and demonstrated that prenatal diagnosis for specific conditions could be achieved within a short period of time with unpurified DNA and nonradiolabelled probes. Several novel approaches to molecular diagnosis had been established. The PCR amplified samples could be analyzed by standard RFLPs, whether or not the mutation altered a restriction site. Sequence polymorphisms could be ascertained with A S 0 probes. This approach would be of considerable value for conditions caused by a number of known mutations. Finally, gene deletions could be ascertained by the failure to detect specific sequences in dot blots of amplified samples. Alternatively, direct sequencing of samples for the detection of rare disease-producing point mutations was feasible following PCR amplification of single copy genes, as demonstrated for /3-thalassemia (28). Viral detection in cells/tissues has been a cumbersome and imprecise undertaking. The PCR technique has proved to be effective in the identification of HIV-1 in patients seropositive but in whom viral culture had been negative (29). Human papilloma virus has been detected in paraffin blocks of biopsies of cervical dysplasia and invasive carcinoma following PCR amplification
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(30). Early in 1989 PCR made possible the detection of HTLV-I sequences in blood mononuclear cells from a series of patients with multiple sclerosis (31). Residual lymphoma has been detected in specimens that were normal with conventional Southern blots (capable of detecting gene rearrangement when the population of malignant cells is only 1%) and flow cytometry (32). The procedure is adequate to amplify, from paraffin tissue, the junctional region in t(14;18) lymphomas (33). Mutant c-K-ras genes have been detected in most human pancreatic carcinomas following PCR amplification of paraffin blocks (34). The technique has also proved of value to assess patients with chronic myeloid leukemia; in this setting, the search for the aberrant transcript (mRNA) for the bcr/abl hybrid was adapted to the PCR following the conversion of mRNA to cDNA with reverse transcriptase and oligoprimers (35). CONCLUSION This overview has attempted to present a novel facet of molecular biology that is certain to impact on our practice. The topic may appear intimidating or irrelevant at first; yet, this technology offers us a previously unknown soil of exploration. This relatively simple technological advance makes possible scores of studies. Our hospital laboratories are filled with mountains of paraffin blocks; this material can assume a new relevance. Not only can the blocks be retrieved and studied, but also we can now fix hazardous tissue in formalin, knowing well that it can be resurrected for study with the PCR. In the final analysis exciting times lie before us. REFERENCES 1 . Smith HO, Wilcox KW. A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J Mol Biol 1970;51:379-91. 2. Cohen S, Chang AC, Boyer HW, Helling RB. Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci (USA) 1973;70:3240-4. 3. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975;98:503-17. 4. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci (USA) 1977;74:5463-7. 5. Maxam AM, Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci (USA) 1977;74:5604. 6. Gait MJ, Sheppard RC, Rapid synthesis of oligodeoxyribonucleotides:A new solid-phase method. Nucleic Acids Res 1977;4:1135-8. 7 . Kan YW, Dozy AM. Polymorphism of DNA sequence adjacent to human B-globin structural gene: Relationship to sickle mutation. Proc Natl Acad Sci (USA) 1978;75:5631-5. 8. Botstein D, White RL, Skolnick M , Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 1980;32:314-31. 9. Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalysed chain reaction. Methods Enzymol 1987;155:335-50.
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10. Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of B-globin genomic sequences and
restriction site analysis for diagnosis of sickle cell anemia. Science 1985;230:1350-4. 11. Lench N, Stanier P, Williamson R . Simple non-invasive method to obtain DNA for gene analysis.
Lancet 1988;1:1356-8.
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12. Higuchi R , von Beroldingen CH, Sensabaugh GF, Erlich HA. DNA typing from single hairs. Nature 1988;332:543-6. 13. Paabo S. Molecular cloning of Ancient Egyptian mummy DNA. Nature 1985;314:644-5. 14. Li H , Gyllensten UB, Cui XF, Saiki RK, Erlich HA, Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 1988;335:414-7. 15. Liidecke HJ, Senger G , Claussen U, Horsthemke B. Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature 1989;338:348-50. 16. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction. Cold Spring Harbor Symp Quant Biol 1986;51:263-73. 1 7 . Oste C . Polymerase chain reaction. Biotechniques 1988;6:162-7. 18. Erlich HA, Gelfand DH, Saiki RK. Specific DNA amplification. Nature 198;331:461-2. 19. Marx JL. Multiplying genes by leaps and bounds. Science 1988;240:1408-10. 20. Saiki RK, Bugawan T L , Horn GT, Mullis KB, Erlich HA. Analysis of enzymatically amplified Bglobin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature 1986;324:163-6. 21. Chien A, Edgar DB, Trela JM. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 1976;127:1550-7. 22. Kogan SC, Doherty M, Gitschier J. An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. N Engl J Med 1987;317:985-90. 23. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988;239:487-91. 24. Scharf SJ, Horn GT, Erlich HA. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science 1986;233:1076-8. 25. Embury SH, Scharf SJ, Saiki RK, et al. Rapid prenatal diagnosis of sickle cell anemia by a new method ofDNA analysis. N Engl J. Med 1987;316:656-61. 26. Impraim CC, Saiki RK, Erlich HA, Teplitz RL. Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides. Biochem Biophys Res Commun 1987;142:710-6. 27. Bos JL, Fearon ER, Hamilton ST, et al. Prevalence of ras gene mutations in human colorectal cancers. Nature 1987;327:293-7. 28. Wong C, Dowling CE, Saiki RK, Higuchi RG, Erlich HA, Kazazian H H J . Characterization of Bthalassaemia mutations using direct genomic sequencing of amplified single copy DNA. Nature 1987;330:384-6. 29. Ou CY, Kwok S, Mitchell SW et al. DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science 1988;239:295-7. 30. Shibata DK, Arnheim N, Martin WJ. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J Exp Med 1988;167:225-30. 31. Reddy EP, Sandberg-Wollheim M, Mettus RV, Ray PE, DeFreitas E, Koprowski H . Amplification and molecular cloning of HTLV-I sequences from DNA of multiple sclerosis patients. Science 1989;24:529-33. 32. Lee MS, Chang KS, Cabanillas F, Freireich EJ, Trujillo J M , Stass SA. Detection of minimal residual cells carrying the t( 14;18) by DNA sequence amplification. Science 1987;237:175-8. 33. Pezzella F, Gatter KC, Mason DY. Detection of 14;18 chromosomal translocation in paraffinembedded lymphoma tissue. Lancet 1989; 1:779-80. 34. Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, Perucho M. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 1988;53:549-54. 35. Morgan GJ, Hughes T, Janssen JW, et al. Polymerase chain reaction for detection of residual leukemia. Lancet 1989;1:928-29.
Fetal Pediatr Pathol Downloaded from informahealthcare.com by York University Libraries on 12/30/14 For personal use only.
POLYMERASE CHAIN REACTION
Thomas A. Seemayer, MD 0 The Nicolas W. Matossian Molecular Pathology Laboratory, The Montreal Children’s Hospital, McGill University Faculty of Medicine, Montreal, Quebec H3H 1P3, Canada
INTRODUCTION Over the past several decades, molecular biology has assumed a position in the forefront of medical science. Nowadays many of the advances in medicine have a decided molecular slant. These contributions have been made possible by fundamental technological and conceptual breakthroughs, which have facilitated the dissection of molecular events central to both health and disease. Among many of these advances, several landmarks come to mind. Not surprisingly, these advances are linked, since one discovery often facilitated those that followed. I n 1970 restriction endonucleases were identified in bacteria (1). The significance of this discovery (akin to the elucidation of the structure of DNA itself) was recognized immediately, for scientists could thereby utilize minute quantities of these enzymes to cleave DNA with precision, thereby generating a multitude of defined segments. This labor led to the birth of molecular cloning, which became a reality in 1973 (2). It then became possible to insert and ligate, virtually at will, small defined DNA segments into bacterial plasmids or phages to produce copious (in molecular terms) amounts of highly purified DNA. For those working (at that time) with DNA in agarose gels, the report of Southern in 1975 describing a simple method for transferring DNA from agarose to filters surely represented a major contribution (3). This procedure also reduced the DNA requirement for most experiments about tenfold over previous methods. The long-standing technical obstacles to rapid and efficient DNA sequencing were overcome by 1977 (4,5 ) . That year, short segments of DNA were synthesized chemically (6); with this, a new term entered the English language: the “gene machine.’’ In 1978, the Address reprint requests to Thomas A. Seemayer, M.D., Department of Pathology, The Montreal Children’s Hospital, 2300 Tupper Street, Montreal, Quebec H3H 1P3, Canada. Pediatric Pathology, 10.311-31 7, 1990 Copyright 0 1990 by Hemisphere Publishing Corporation
31 1
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312
T. H. SEEMAYER
discovery of DNA-sequence polymorphisms adjacent to the human P-globin structural gene (7) set the stage for the broad application of restriction fragment length polymorphisms (RFLPs) (8) as molecular genetic markers for a vast assortment of diseases. These collective advances made possible the analysis of molecular aberrations central to many human diseases. Early on, the hemoglobinopathies were a prime target of investigation. More recently, diseases such as cystic fibrosis, Duchenne muscular dystrophy, Huntington’s disease, Alzheimer’s disease, schizophrenia, and diverse neoplasms are being understood in a manner never before possible. For the most part these studies require the extraction, purification, electrophoretic separation, and cloning of genomic DNA from blood lymphocytes, chorionic villi, or fragments of fresh or frozen tissue. Clearly the process worked and at the time was considered ideal. Now, however, molecular analysis and cloning call for an amount of source DNA (5-10 mg) and labor (days to weeks) well in excess of that required in light of further technological development.
OVERVIEW OF THE POLYMERASE CHAIN REACTION In 1985, a new and revolutionary procedure, the polymerase chain reaction (PCR), was developed by Mullis and colleagues at the Cetus Corporation (9, 10). As is often the case with discoveries of this magnitude, the principles are relatively simple. The description of this procedure and its applications follow. The PCR, an in-vitro method for the primer-directed enzymatic amplification of specific DNA sequences, mimics normal DNA replication in that each cycle of the reaction results in the doubling of DNA. The process allows for the generation of million of copies of a highly specific (usually short, e.g., 15-400 base pair) DNA segment within hours. The source of DNA to be amplified can be provided by fresh or frozen tissue, blood lymphocytes, chorionic villi, buccal epithelial cells (1 l), hair follicles (12), and even formalinfixed tissue within paraffin blocks. Because DNA has been recovered and cloned from a radiocarbon dated 2,400-year-old Egyptian mummy (1 3), the potential applications of PCR amplification in the study of archival specimens appears limitless. The quantity of DNA required for amplification is infinitesimal: a single hair root with a follicle, a single diploid cell, a single sperm (14), a drop of blood-even a microdissected region from a single chromosome (15) can serve as source material. The ability of the PCR to amplify such minute amounts of target, however, demands that the strictest care be employed to avoid contamination with DNA of an extraneous source. The DNA need not
Fetal Pediatr Pathol Downloaded from informahealthcare.com by York University Libraries on 12/30/14 For personal use only.
POLYMERASE CHAIN REACTION
313
be “in good shape,” that is, purified or of high molecular weight; partiallydegraded DNA will provide a suitable target for amplification provided that there is a sufficient amount of single-stranded DNA to bridge the distance between the 5’ ends of the two primers. Any segment of interest, even a single copy gene buried cryptically amidst the entire human genome, can be amplified. Not only does the procedure work with DNA as source material (either single- or double-stranded), but it also can be applied to RNA. With a minor modification, RNA, when expressed, can be converted to firststrand cDNA with reverse transcriptase and oligoprimers, and amplification can proceed. Because of the magnitude of amplification, radiolabelled probes can be avoided; the amplified segment can be visualized with ethidium bromide (in agarose gels) or nonradiolabelled probes. With a recent refinement of the procedure, the process has been automated. In the end, picograms of source DNA (target and nontarget) are amplified to microgram amounts (target) within several hours. Finally, since amplification is of this level of magnitude, the need for subcloning can be obviated, and the fragments can be sequenced directly. The essence of the PCR resides in the selection of a set (or two sets in the case of “nested”primers) of oligonucleotide primers complementary to a portion of the coding sequence of the segment to be amplified. These primer nucleotide sequences (usually about 20) are synthesized chemically. The oligoprimers flank the segment to be amplified, anneal to their complementary DNA sequences, and direct DNA synthesis in the 5‘ to 3 ‘ direction. Typically, the primers are not complementary to one another; supplied in excess relative to the target DNA template, the formation of primer-template complexes (synthesized in opposite and overlapping directions) is favored over the reassociation of the two DNA strands following the later reduction of temperature. With each cycle, the amount of newly synthesized DNA is doubled, because the extension product of one primer functions as the template for the other. The end of the specific fragment produced is determined by the 5 ’ end of the oligoprimers (which have become incorporated into the product). Although the 5’ end is usually matched to the original template, it may be purposely mismatched, thereby allowing the introduction of other sequences into the product. Thus, insertions, deletions, nucleotide substitutions (in-vitro mutations), regulatory elements, and even restriction site linkers (for later linkage to cloning vectors for sequencing) can be added. With repeated cycles, two DNA products are produced. The “short product,” the region between the 5 ’ ends of the extension primer, is,a discrete, double-stranded DNA molecule produced in great excess. This molecule is of interest because its defined ends correspond to the primers’ sequence. A “long product,” derived from the template molecules in each cycle, is produced in far lesser amounts and is characterized by variable nucleotides at the 3 ’ end (1 6- 19).
314
T. H. SEEMAYER
Fetal Pediatr Pathol Downloaded from informahealthcare.com by York University Libraries on 12/30/14 For personal use only.
THE REACTION The reaction proceeds in three steps (which constitute a cycle), which are repeated over many (20 or more) cycles. The first step, denaturation, brought about by high temperature, separates the two strands of the target DNA molecule. The second, annealing of the extension primers to the DNA template, takes place at a lower temperature (37"-55"C). The third, primer extension with deoxynucleotide triphosphates and DNA polymerase, is performed at a higher temperature (72°C). In the original descriptions (9, 10, 20), the Klenow fragment of E. coli DNA polymerase I was employed at 37 "C. The process was tedious and expensive, because this fragment was thermolabile and inactivated following the heat denaturation step; thus, fresh enzyme had to be added with each cycle. As well, a variety of products was produced, only some of which were specific. In 1987-1988 the process was improved following the substitution of a thermostable DNA polymerase derived from a thermophilic bacterium, Themus aquaticus (Tag) (2 1). This polymerase, usually added only once at the time of the first cycle, is stabile under denaturation conditions and results in a markedly enhanced yield of specific-sized fragments. Single copy sequences have been amplified over 10 millionfold with very high specificity. Moreover, the method has the capability to amplify and detect a target DNA molecule contained in a sample of 100,000 cells. In addition, long specific amplified fragments have been produced (up to 3.2 kilobases) (22, 23). The superiority of this polymerase led directly to the development of a DNA Thermal Cycler (Perkin-Elmer Cetus Instruments), thereby automating the PCR.
POLYMERASE CHAIN REACTION APPLICATIONS The first report of PCR amplification included a clever technique to differentiate between the A and S alleles in the beta chain of human hemoglobin. Following PCR amplification (some 200,000 times) of target DNA, oligomer restriction analysis was performed (10). In this maneuver, the amplified DNA was hybridized to a labelled oligonucleotide probe and restriction enzyme cleavage of the duplex was performed. Because of a designed mismatch within the restriction site in the sickle variant of the beta globin gene and the probe, cleavage did not take place; thus the distinction between the two forms of the hemoglobin molecule was a relatively simple task following electrophoresis and radioautography. The second application established that PCR amplification could be followed by the direct cloning and sequencing of amplified genomic DNA. This advance was brought about with an oligoprimer modification at one of the 5 '
Fetal Pediatr Pathol Downloaded from informahealthcare.com by York University Libraries on 12/30/14 For personal use only.
POLYMERASE CHAIN REACTION
315
ends to produce restriction site linkers. In this report, a 110-base pair fragment of the human P-globin gene and a 242-base pair fragment of the HLAD Q alpha locus were analyzed (24). PCR amplification followed by dot blot analysis with allele-specific oligonucleotide probes (ASO) came next. This is a particularly elegant utilization of the PCR, one with countless applications. These short probes are specific to the extent that a single base pair mismatch negates probe-target recognition (binding). Following the amplification of a 110 bp fragment of the sixth codon of the human 0-globin gene, specific oligoprobes for hemoglobin A, S, and C easily distinguished the hemoglobin content in the samples (20, 25). The amplification was also shown to be effective with crude cell lysates, thereby negating the requirement for DNA purification. As a follow-up, the procedure worked well with DNA extracted from paraffin blocks (26). A similar approach reported about this time employed frozen tissue and oligoprobes to establish the high incidence of c-Ki-ras mutations at codon 12 in colorectal carcinomas (2 7). The first description of Taq polymerase-directed PCR’s came in a study of blood (finger pricking) and chorionic villi biopsies in a setting of hemophilia A focused on carrier detection and prenatal diagnosis (22). When the amplified products were digested, appropriately sized fragments were visualized in the ethidium bromide treated gels. Allele-specific oligoprobes in dot blots were able to distinguish a single base-pair alteration responsible for a BclI polymorphism. Fetal sex was established by amplification of the Y-chromosome 3.4 kb repeats. This study (and one that soon followed) (23) established the superiority of Taq polymerase to the Klenow fragment and demonstrated that prenatal diagnosis for specific conditions could be achieved within a short period of time with unpurified DNA and nonradiolabelled probes. Several novel approaches to molecular diagnosis had been established. The PCR amplified samples could be analyzed by standard RFLPs, whether or not the mutation altered a restriction site. Sequence polymorphisms could be ascertained with A S 0 probes. This approach would be of considerable value for conditions caused by a number of known mutations. Finally, gene deletions could be ascertained by the failure to detect specific sequences in dot blots of amplified samples. Alternatively, direct sequencing of samples for the detection of rare disease-producing point mutations was feasible following PCR amplification of single copy genes, as demonstrated for /3-thalassemia (28). Viral detection in cells/tissues has been a cumbersome and imprecise undertaking. The PCR technique has proved to be effective in the identification of HIV-1 in patients seropositive but in whom viral culture had been negative (29). Human papilloma virus has been detected in paraffin blocks of biopsies of cervical dysplasia and invasive carcinoma following PCR amplification
Fetal Pediatr Pathol Downloaded from informahealthcare.com by York University Libraries on 12/30/14 For personal use only.
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(30). Early in 1989 PCR made possible the detection of HTLV-I sequences in blood mononuclear cells from a series of patients with multiple sclerosis (31). Residual lymphoma has been detected in specimens that were normal with conventional Southern blots (capable of detecting gene rearrangement when the population of malignant cells is only 1%) and flow cytometry (32). The procedure is adequate to amplify, from paraffin tissue, the junctional region in t(14;18) lymphomas (33). Mutant c-K-ras genes have been detected in most human pancreatic carcinomas following PCR amplification of paraffin blocks (34). The technique has also proved of value to assess patients with chronic myeloid leukemia; in this setting, the search for the aberrant transcript (mRNA) for the bcr/abl hybrid was adapted to the PCR following the conversion of mRNA to cDNA with reverse transcriptase and oligoprimers (35). CONCLUSION This overview has attempted to present a novel facet of molecular biology that is certain to impact on our practice. The topic may appear intimidating or irrelevant at first; yet, this technology offers us a previously unknown soil of exploration. This relatively simple technological advance makes possible scores of studies. Our hospital laboratories are filled with mountains of paraffin blocks; this material can assume a new relevance. Not only can the blocks be retrieved and studied, but also we can now fix hazardous tissue in formalin, knowing well that it can be resurrected for study with the PCR. In the final analysis exciting times lie before us. REFERENCES 1 . Smith HO, Wilcox KW. A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J Mol Biol 1970;51:379-91. 2. Cohen S, Chang AC, Boyer HW, Helling RB. Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci (USA) 1973;70:3240-4. 3. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975;98:503-17. 4. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci (USA) 1977;74:5463-7. 5. Maxam AM, Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci (USA) 1977;74:5604. 6. Gait MJ, Sheppard RC, Rapid synthesis of oligodeoxyribonucleotides:A new solid-phase method. Nucleic Acids Res 1977;4:1135-8. 7 . Kan YW, Dozy AM. Polymorphism of DNA sequence adjacent to human B-globin structural gene: Relationship to sickle mutation. Proc Natl Acad Sci (USA) 1978;75:5631-5. 8. Botstein D, White RL, Skolnick M , Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 1980;32:314-31. 9. Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalysed chain reaction. Methods Enzymol 1987;155:335-50.
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