The Polymerase Chain Reaction JOY D. JESTER, MD

he Polymerase Chain Reaction (PCR) technique has proved to be a major advance in the detection of specific nucleic acid fragments. It is a newly described technique that allows for the selective amplification of discrete DNA sequences amidst a larger body of DNA. Using the l?CR, DNA that was initially present in a minute quantity may be specifically amplified to a readily detectable level. The technique was developed and first described by Mullis and associates and is essentially modeled after the natural DNA replication process.1-6 The development of the thermostable DNA polymerase purified from Thermus aquaticus (Ta9 DNA Polymerase) and Cetus’ DNA Thermal Cycler machine helped automate the process of gene amplification and therefore made the technique more amenable to clinical applications, The process of the PCR involves the repetition of three basic steps: denaturation, annealing, and extension. These steps are outlined in Figure 1. Denaturation of the native double-stranded DNA (called the template) occurs at a high temperature. Once dissociated the two strands remain free in solution until the temperature is low enough to permit annealing. The primers consist of two separate oligonucleotides that anneal to DNA sites flanking the sequence of interest. The primers are present in excess of the template DNA in the mixture so that the template preferentially anneals to primers rather than reannealing to its opposite strand. Annealing of the primers to the template strand occurs at a lower temperature; the solution is then heated to allow extension of the primers. Initially, in the presence of an excess of nucleotide building blocks, the DNA polymerase enzyme ex-

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From the Depnrtment of Dermatology, University of Colorado Health Sciences Center, Denver, Colorudo. Address correspondence to: joy D. fester, M.D., Assistant Professor, Department of Pathology and Department of Medicine, Division ofDenutology, Vunderbilt University, 3990 The Vanderbilt Clinic, Nashville, TN 37232.

0 1991 by Elsevier Science Publishing

Co., Inc.

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0738-081x/91/$3.50

tends the primer sequence using the single strand of sample DNA as a template. After several cycles have passed, the “short product”, ie, the region between the 5’ ends of the primers, is predominant in the reaction mixture and becomes the dominant template to which primers anneal. Theoretically the amount of short product doubles after each cycle so that the end accumulation of the target fragment approaches 2”, where n is the number of cycles. DNA may be extracted from both fresh frozen and paraffin-embedded specimens of ~kin.‘~~ Generally, about a l-mm? fragment of a frozen biopsy or about ten to twenty 5-pm sections of a paraffin block are sufficient. The primers are synthesized oligonucleotides that flank the DNA sequence of interest. Selection of new primers can be difficult; generally, computer programs are utilized to determine the presence of homologous sequences that could self anneal to either form hairpin loops within a primer or cause the two primers to anneal to each other. The DNA extracted from the specimen is added to a mixture that includes reaction buffer, nucleotide bases, the two primers, and the Ta9 Polymerase enzyme. The reaction tube is then placed in the cycler unit, which has been programmed to cycle through the temperatures required for repetitive denaturing, annealing, and extension. Amplified samples are generally run on acrylamide gels along with positive controls. Bands in the sample lanes can be compared to the positive control by position on the gel (which corresponds to molecular weight). To further verify the specificity of the bands some labs transfer the DNA to nitrocellulose filters and perform Southern blots with a radionucleotide-labeled internal oligonucleotide corresponding to a segment between the two primers9 (Figure 2). It should be noted that conditions necessary for successful amplification vary depending on the sequence sought. In the author’s experience some segments amplify with a wide range of annealing temperatures, template concentrations, and buffer content, whereas other

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The role of human papilloma virus (HPV) in human disease is another area of current research activity. HPV DNA has been identified in dysplastic and malignant lesions of the conjunctiva and cornea*4 and in cervical cancer tissue15 by PCR. The recent development of primers to conserved sequences of HPV DNA will greatly ease screening of tissue for the presence of HPV.16 For example, a recent study examined biopsies of trichilemmonas for the presence of HPV and could not demonstrate the virus in 25 samples.” In addition to providing a rapid means of determining a cutaneous infection with herpes simplex virus (HSV),‘* PCR has been used to verify the association of HSV with various skin diseases. HSV DNA was demonstrated in approximately two-thirds of all biopsies of recurrent erythema multiforme whether they were clinically associated with herpetic infections or not.19 The author is currently investigating the role of HSV in other diseases such as aphthous stomatitis,20 as well as the presence of HSV in the peripheral blood of normal patients with acute herpes labialis. Various other viruses are currently under investiga-

DNA TEMPLATE SEl2”ENCEOFlNTEREST A f 1 5 3

1. DENATURATION (HIGHER

2. ANNEALING (LOWER

TEMPERATURE)

+ PRIMERS

+ Taq + dNTPs

OF PRIMERS TEMPERATURE)

3. EXTENSION OF PRIMERS

MULTIPLE CYCLES OF THESE THREE STEPS YIELDS A GEOMETRIC INCREASE IN THE DNASEQUENCE OF INTEREST SO IT BECOMES THE PREDOMINANT SPECIES PRESENT

I=-

-

F-”

-Figure

1.

An outline of the basic steps involved in PCR.

DNA segments are fastidious in their requirements. ditions must be adjusted for each system.

Con-

Applications of PCR to Dermatopathology The PCR technique has been particularly effective in demonstrating the presence of infectious agents in tissue. Many organisms can be difficult and laborious to culture or demonstrate in tissue. PCR can supplement cumbersome techniques to provide a rapid verification of the presence of an organism and probably will become a routine clinical test for certain infectious agents. The study of viruses has benefited greatly from the use of PCR, especially concerning human immunodeficiency virus type I (HIV-l). PCR has been used to demonstrate the presence of HIV-l in the blood of individuals who are sero-negative, such as silent infections in which the patient has reverted from seropositive to seronegative,‘O individuals who have been exposed to the virus but have and infants of HIV-positive not yet seroconverted,” mothers.12 Another report has shown that all study subjects who were seropositive had detectable HIV-l in their blood, lending support to the premise that antibody positivity correlates with a present HIV infection.13

Figure 2. An example of an autoradiograph of a Southern analysis of DNA amplified by PCR. The first lane in each pair represents DNA amplified with primers for the human /3-globin gene, which is ubiquitous in human tissues. The second lane represents DNA amplified with primers for a segment of the herpes simplex genome. The first pair of lanes demonstrates the results with a specimen of herpes labialis, the second lane a biopsy of chronic dermatitis, the third a biopsy of herpesassociated e ythema multiforme, and the last lane a biopsy of idiopathic eyfkema mulfiforme. All specimens amplified for the presence of &$obin whereas only biopsies of herpes labialis and e ythema multiforme amplified for herpes simplex virus DNA.

HSV

NEG

HAEM

IPEM

Clinics in Dermatology 1991;9:137-141 tion using the PCR technique, including cytomegaloviru~,~*-~~Epstein-Barr virus,8 human parvovirus,s Hepatitis B viru~,~~and enteroviruses.27 Recently the presence of HTLV-1 was demonstrated in a small percentage of biopsies of mycosis fungoides2* In addition to viruses PCR may be useful to diagnose the presence of other infective organisms such as Toxo-

plasma gondii,2g Trypanosoma cruzi,30 Mycoplasma pneumoniae,31 and Legionella pneumophila.32 Of great interest to dermatologists will be the development of PCR screening for diseases with cutaneous findings. A PCR assay for the Lyme Disease spirochete Borrelia burgdorferi has recently been described. 33 Such a clinical test could prove invaluable as the organism is notoriously difficult to culture and demonstrate in tissue. The early diagnosis of Rocky Mountain Spotted Fever has often depended on direct immunofluorescence-antibody tests of skin biopsies to show the presence of Rickettsia ricketfsii. Two groups have reported detecting Rickettsia using I’CR.34,35 The identification of Mycobacterium can be time-consuming given the long culture time required for the organisms. Pate1 et al have used PCR to rapidly identify the presence of M. tuberculosis.36 Whereas most of the tests described above are currently available only in research labs, clinical labs will doubtless rely on PCR more and more as the applications become routine. PCR for some viral agents have already become routine in some medical centers. As such small amounts of material are necessary for analysis, PCR is already widely used in forensic pathology to identify the individual source of certain samples such as human sperm. 37 For similar reasons PCR promises to greatly aid HLA typing and tissue typing for organ transplantation. 38,3gAnother important area of application is the prenatal diagnosis of genetic disorders. Hemophilia A was one of the first diseases detected prenatally with l?CR.40 Many other diseases are amenable to this, particularly diseases with restriction-site variation, such as sickle cell anemia41 and Duchenne’s muscular dystrophy,42 as well as gene deletions and mutations such as thalassemia.43,44 Other diseases identified prenatally include Huntington’s disease,45 Lesch-Nyan syndrome,“j and cystic fibrosis.” The PCR has proved very useful in screening individual patients for residual tumor burden, predominantly leukemia4a and lymphoma4g at present. Of interest to dermatologists is a recent abstract describing the development of a probe specific for a patient’s malignant T-lymphocytes that enabled clinicians to follow the presence of residual disease in a patient with Sezary’s syndrome.50 The technique has been invaluable in investigations of alleles and their part in the pathogenesis of disease. This is particularly true of studies involving oncogenes and

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cancer such as acute myeloid leukemia,5’ pancreatic carcinoma, and colorectal carcinoma.53 Autoimmune-linked HLA alleles have also been identified in patients with diabetes mellitus,54 myasthenia gravis,55 multiple sclerosis,55 and pemphigus vulgaris.56

Disadvantages of the Technique From the viewpoint of a histopathologist, a major drawback of the technique is that it does not allow localization of the DNA to specific structures within a specimen. In situ hybridization is an attractive companion to the PCR technique to allow this localization. Another disadvantage of the technique is the need for synthesis of specific primers. Not only is the process costly, determination of the proper primer sequences can be difficult. This problem is likely to be of less importance in microbiology labs, where many samples may be screened for a given infectious agent and volume will bring down the cost of the procedure. The most important shortcoming of the PCR technique is the high probability of contamination. The technique is so sensitive that contaminants become a major source of false positives. Careful handling of all specimens is imperative and sample specimens should not manipulated in close proximity to positive controls and amplified specimens. Positive displacement pipettes are very helpful to avoid splash contamination while pipetting. Basic reaction mixtures should be dispensed with separate DNAfree pipettemen and should be stored separately from DNA-containing specimens. Especially when dealing with experiments identifying small quantities of DNA that may be latent in skin it may be necessary to maintain separate extractions of a given specimen to insure that the specimen itself has not been contaminated. Repeating reactions helps assure that positive results are real and not a result of contamination during the process.57,58

Summary In a short time the PCR techniques has revolutionized research technology in many areas of medicine. Because of the ease and rapidity of the technique it is quickly becoming a standard clinical test for many diseases. Clinical applications continue to emerge from research labs and should rapidly expand to facilitate rapid medical diagnosis.

References 1. Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase catalysed chain reaction. Meth Enzymol 1987;155:335-50.

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2. Saiki RK, Scharf S, Faloona KB, et al. Enzymatic amplification of P-globin genomic sequence and restriction site analysis for diagnosis of sickle cell anemia. Science 1985;230:1350-54. 3. Oste C. Polymerase 1988;6(2):162-67.

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Biotechniques

4. Landegren U, Kaiser R, Casey CT, Hood L. DNA diagnostics-Molecular techniques and automation. Science 1988;242:229-37. 5. Eisenstein BI. The polymerase chain reaction. N Engl J Med 1990;322(3):178-83. 6. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988;239:487-91. 7. Shibata DK, Amheim N, Martin WJ. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction, J Exp Med 1988;167:225-30. 8. Abbott MA, Poiesz BJ, Byrne BC, et al. Enzymatic gene amplification: Qualitative and quantitative methods for detecting proviral DNA amplified in vitro. J Infect Dis 1988;158(6):1158-69. 9. Sixbey JW, Shirley I’S Viral diagnosis using DNA-based probes-enzymatic amplification of target DNA. Ann NY Acad Sci 1988;549:158-65. 10. Loche M, Mach B. Identification of HIV-infected seronegative individuals by a direct diagnostic test based on hybridisation to amplified viral DNA. Lancet 1988:ii;418-21. 11. Imagawa DT, Lee MH, Wolinsky SM, et al. Human immunodeficiency virus type I infection in homosexual men who remain seronegative for prolonged periods. N Engl J Med 1989;320:1458-62. 12. Rogers MF, Ou C-Y, Rayfield M, et al. Use of the polymerase chain reaction for early detection of the proviral sequences of human immunodeficiency virus in infants born to seropositive mothers. 1989;320:1649 - 1654. 13. Jackson JB, Kwok SY, Sninsky JJ, et al. Human immunodeficiency virus type I detected in all seropositive symptomatic and asymptomatic individuals. J Clin Microbial 1990;28(1):16-19.

19. Brice SL, Krzemien D, Weston WL, Huff JC. Detection of herpes simplex virus DNA in cutaneous lesions of erythema multiforme. J Invest Dermatol 1989;93(1):183-87. 20. Jester JD, Stockert SS, Huff JC, Brice SL. Detection of herpes simplex virus DNA in lesions of recurrent aphthous stomatitis using the polymerase chain reaction (abstr). J Invest Dermatol 1990;94(4):538. 21. Brice SL, Stockert SS, Jester JD, et al. Detection of herpes simplex virus DNA in peripheral blood cells during acute herpes labialis using the polymerase chain reaction (abstr) J Invest Dermatol 1990;94(4):509. 22. Shibata D, Martin WJ, Appleman MD, et al. Detection of cytomegalovirus DNA in peripheral blood of patients infected with human immunodeficiency virus. J Inf Dis 1988;158(6):1185-92. 23. Demmler GJ, Buffone GJ, Schimbor CM, May RA. Detection of cytomegalovirus in urine from newborns by using polymerase chain reaction DNA amplification. J Infect Dis 1988;158:1177-84. 24. Cassol SA, Poon MC, Pal R, et al. Primer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA: Application to the early diagnosis of CMV infection in marrow transplant recipients. J Clin Invest 1989;83:1109-15. 25. Clewley JP. Polymerase chain reaction assay of parvovirus B19 DNA in clinical specimens. J Clin Microbial 1989;27(12):2647-51. 26. Ulrich PI’, Bhat RA, Seto B, et al. Enzymatic amplification of hepatitis B virus DNA in serum compared with infectivity testing in chimpanzees. J Infect Dis 1989;160:37-43. 27. Chapman NM, Tracy S, Gauntt CJ, Fortmueller U. Molecular detection and identification of enteroviruses using enzymatic amplification and nucleic acid hybrids. J Clin Microbiol 1990;28(5):843-50. 28. Whittaker SJ, Smith NP, Russell Jones R, Luzzatto L. HTLV-I and cutaneous T-cell lymphoma (abstr). J Invest Dermatol 1990;94(4):590. 29. Burg JL, Grover CM, Pouletty I’, Boothroyd JD. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. J Clin Microbial 1989;27:1787-92.

14. McDonnell JM, Mayr AJ, Martin WJ. DNA of human papillomavirus type 16 in dysplastic and malignant lesions of the conjunctive and cornea. N Engl J Med 1989;320(22):144246.

30. Moser DR, Kirchhoff LV, Donelson JE. Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction. J Clin Microbial 1989;27:1477-82.

15. Yu-kai H, Ji-zeng Z, Qian X, Ji-ming G. Detection of human papillomavirus DNA in cervical cancer tissue by the polymerase chain reaction. J Virol Methods 1989;26:17-26.

31. Bernet C, Garret M, DeBarbeyrac B, et al. Detection of Mycoplasma pneumoniae by using the polymerase chain reaction. J Clin Microbial 1989;27(12):2492-96.

16. Gregoire L, Arella M, Campione-Piccardo J, Lancaster WD. Amplification of human papillomavirus DNA sequences by using conserved primers. J Clin Microbial 1989;27(12): 2660-65.

32. Stambach MN, Falkow S, Tompka LS. Species-specific detection of Legionella pneumophila in water by DNA amplification and hybridization. J Clin Microbial 1989;27:125761.

17. Leonardi CL, Zhu WY, Penneys NS, Kinsey WH. Trichilemmomas are not associated with human papillomavirus DNA (abstr). J Invest Dermatol 1990;94(4):548.

33. Rosa PA, Schwan TG. A specific and sensitive assay for the Lyme Disease spirochete Borrelia burgdorferi using the polymerase chain reaction. J Inf Dis 1989;160(6):101829.

18. Cao M, Xiao X, Egbert B, et al. Rapid detection of cutaneous herpes simplex virus infection with the polymerase chain reaction. J Invest Dermatol 1989;92:391-92.

34. Tzianabos T, Anderson B, McDade J. Detection of Rickeffsia rickeffsia DNA in clinical specimens by using polymerase

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36. Pate1 RJ, Fries JWU, Piessens WF, Wirth DF. Sequence analysis and amplification by polymerase chain reaction of a closed DNA fragment for identification of Mycobacterium tuberculosis. J Clin Microbial 1990;28(3):51318.

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46. Gibbs RA, Nguyen PN, McBride LJ, et al. Identification of mutations leading to the Lesch-Nyan Syndrome by automated direct DNA sequencing of in vitro amplified cDNA. Proc Nat1 Acad Sci USA 1989;86:1919-23. 47. Williams C, Weber L, Williamson R, Hjelm M. Guthrie Spots for DNA-based carrier testing in Cystic Fibrosis. Lancet 1988;ii:693. 48. Morgan GJ, Janssen JWG, Guo A, et al. Polymerase chain reaction for detection of residual leukaemia. Lancet 1989;i:928-29.

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51. Farr CJ, Saiki RK, Erlich HA, et al. Analysis of RAS gene mutations in acute myeloid leukemia by polymerase chain reaction and oligonucliotide probes. Proc Nat1 Acad Sci USA 1988;85:1629-33. 52. Almoguera C, Shibata D, Forrester K, et al. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 1988;53:549-54. 53. Bos JL, Fearon ER, Hamilton SR, et al. Prevalence of ras gene mutations in human colorectal cancers. Nature 1987;327:293-97. 54. Miyano M, Nanjo K, Chan SJ, et al. Use of DNA Amplification to screen family members for an insulin gene mutation. Diabetes 1988;37:862-66. 55. Oksenberg JR, Sherritt M, Begovich AB, et al. T-cell receptor V, and C, alleles associated with Multiple Sclerosis and Myasthenia Gravis. Proc Nat1 Acad Sci USA 1989;86:98892.

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The polymerase chain reaction.

In a short time the PCR techniques has revolutionized research technology in many areas of medicine. Because of the ease and rapidity of the technique...
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