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PCR and molecular pathology
PCR and molecular pathology
D. M. Sexton, M. A. Bennett and D. T. Croke
Introduction The aetiology, pathogenesis and prognosis of human disease are routinely assessed by a variety of biochemical and immunohistochemical techniques. However, in recent years an expanding array of molecular biological techniques have increasingly been used as adjuncts to these, and have yielded substantial information contributing to our understanding of the genetic changes which occur during disease progression. Molecular biology is essentially the study of cellular genetic material (DeoxyriboNucleic AcidIRiboNucleic Acid (DNA/RNA)). DNA, tightly packed with proteins, condenses to form chromosomes just before cell division. DNA exists in a highly ordered double helical conformation, consisting of two linear and antiparallel nucleic acid strands. Each strand consists of an alternating sugar-phosphate backbone to which organic bases are attached. The backbone confers structural rigidity on the molecule, while the genetic information is encoded by the sequence of bases. Four possible bases can be incorporated into the DNA molecule, adenine (A), guanine (G), cytosine (C) and thymine (T). Base pairing between the two antiparallel strands follows a set pattern in which A always pairs with T and C always pairs with G. This is referred to as complementarity. A sequence of DNA which encodes a single protein is referred to as a gene. Structurally, eucaryotic genes consist of regions coding for protein, called exons, which are separated by noncoding intervening sequences referred to as introns. DNA in the cell of any one human individual contains information for about 100 000 genes, only about 15% of which are expressed in a specific cell type, conferring defined structural and functional
D. M. Sexton, M. A. Bennett and D. T. Croke, Department of Biochemistry, Royal College of Surgeons in Ireland, 123, St Stephens Green, Dublin 2, Ireland
properties on the cell. An expressed protein is transcribed from DNA to an intermediate known as messenger RNA (mRNA). Base triplets (codons) determine the incorporation of amino acids into the newly synthesised protein in the order determined by the original DNA strand. To date the nucleotide sequences of approximately 3000 human genes are known. Rapid progress in the identification and characterisation of gene sequences is being made by the Human Genome Project which hopes to generate a physical map of the entire human genome by the end of this decade. Genetic changes underlie the development of many diseases due to aberrant gene expression. As the nucleotide sequences of genes become known, the elucidation of specific diseasecausing genetic aberrations including point mutations, deletions (loss of gene sequence), or translocations will contribute immensely to our understanding of the pathological characteristics of disease. Pathogenic mutations may occur in exons, introns or regulatory regions of genes. The contribution of a pathological mutation in a single gene to the clinical phenotype is dependent on the dominance/recessivity of the mutant gene in relation to the normal gene, the proportion of mature cells in which the mutant gene is present and in some cases the parental origin of the mutation.
The polymerase chain reaction The most exciting and innovative molecular biology technique developed to date is the PoIymerase Chain Reaction (PCR) which offers immense promise in diagnostic and prognostic applications. PCR represents a powerful in vitro method which, due to its sensitivity, speed and selectivity has superseded previous conventional molecular biological techniques, such as Southern blotting analysis and in situ hybridisation, in biomedical research. The method essentially involves the selective amplification of specific segments of DNA.’ The basic
PCR AND MOLECULAR PATHOLOGY
aquaticus renders the process amenable to automation. Amplification of RNA is also possible but requires a reverse transcription step prior to PCR. The quality and quantity of target nucleic acid required for PCR analysis represent features of the technique that are very attractive to the pathologist. In contrast to conventional molecular biological techniques, much smaller quantities of DNA are required to amplify a specific sequence by PCR. Nanogram or even picogram concentrations of template are sufficient for PCR; conventional molecular biological techniques require micrograms of nucleic acid. High quality DNA can be isolated from fresh or frozen tissue. However, many pathology laboratories possess a vast repertoire of human tissues fixed
reaction, carried out in a programmable thermocycler, involves repetitive cycles of DNA synthesis. Each cycle consists of three steps (Fig. 1). The first step involves denaturation of the target nucleic acid which renders it single stranded. This is followed by annealing of synthetic oligonucleotide primers specifically designed to hybridise to the target nucleic acid region. The third step involves extension from the annealed primers, catalysed by a DNA polymerase enzyme. In a typical PCR analysis 20-40 such cycles are carried out. As successive products are generated these become templates for subsequent cycles resulting in exponential amplification of the target region. The use of the thermostable Taq DNA polymerase from the thermophilic bacterium Thermus Double-stranded
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Fig. l-Diagrammatic representation of the three steps involved in a PCR cycle. Step 1 involves denaturation of the target nucleic acid tb its single stranded form (typically 94°C). In step 2 annealing takes place: oligonucleotide primers hybridise to their complementary regions on the target nucleic acid sequence (37-65°C). In step 3 there is extension of the annealed primers using Taq DNA polymerase (72°C). Repeated cycles of PCR amplification, using a programmable thermocycler, results in a exponential increase in the copy - number of sequence.
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in formalin and embedded in paraffin. Fixation with formalin causes cross-linking between DNA and proteins and results in the DNA becoming more rigid and susceptible to mechanical shearing during extraction and consequently can have a profound effect on the integrity of extracted DNA. Several methods, generally involving proteinase K digestion, have been reported for the extraction of DNA from paraffin-embedded tissue. As PCR does not require a high molecular weight template, the crude and somewhat degraded DNA extracted from paraffin-embedded tissue is amenable to amplification by PCR and fragments of up to 500 base pairs are attainable. The potential to use DNA extracted from archival material facilitates retrospective studies on large numbers of patients, making it possible to chronologically trace the molecular progression of disease. A single 5 urn section from formalin-fixed paraffin-embedded tissue contains enough DNA even after 40 years to serve as a template in PCR.2 The labile nature of RNA and its susceptibility to degradation by ubiquitous ribonuclease enzymes renders its isolation inherently more difficult than that of DNA. A procedure for the isolation of RNA from paraflinembedded tissue based on the standard DNA extraction protocol has been reported.3 More recently a rapid method based on the binding of RNA to acid-treated glass beads in the presence of a high concentration of guanidinium was described.“ RNA amplification products of up to 500 base pairs have been obtained from RNA isolated by this procedure. The efficient isolation of RNA from archival material has immense implications in diagnostic pathology as it provides a means to understand changes in gene expression occurring during disease pathogenesis. The innovative yet simple basic PCR results in the generation of millions of copies of a particular target fragment, which can be used directly in diagnosis or can be subjected to further analysis to reveal detailed information regarding genetic mechanisms in disease pathogenesis. A number of modifications of the original PCR have been developed which allow the identification and characterisation of viral and microbial pathogens, detection of point mutations underlying a range of genetic diseases and analysis of changes in gene expression and of gene rearrangements peculiar to specific pathological processes. The following techniques represent some of the variations of PCR applicable to the investigation of disease, and will enable pathologists to evaluate resected tissues and archival material for a range of genetic abnormalities. Detection of disease causing mutations using polymerase chain reaction Detection of point-mutations underlying genetic disease by PCR technology can be accomplished by a sensitive method termed PCR-Single Strand Conformation Polymorphism (PCR-SSCP) which is based on the ability of a single point-mutation to alter the conformation and consequent migration of single-stranded DNA during
electrophoresis. In PCR-SSCP the target sequence in genomic DNA or cDNA is simultaneously amplified and labelled by the incorporation of radioactive primers or nucleotides which allow subsequent detection of the DNA fragment by autoradiography. Following amplification, the double-stranded PCR product is denatured to its single-stranded form and subjected to non-denaturing gel electrophoresis. The conformation (folding pattern) of the single-stranded DNA, which is dictated by the nucleotide sequence, determines its electrophoretic mobility. Assuming that all base changes alter conformation, the presence of a mutation is reflected in a comparative mobility shift between a mutant and a wildtype sequence. The sensitivity of PCR-SSCP in detecting point mutations, which can reach up to 1OO%,5is dependent on several factors including fragment length, gel constituents and temperature of electrophoresis. This technique facilitates screening for activating mutations in oncogenes, inactivating mutations in tumour suppressor genes and underlying mutations in inherited disorders. While this technique is sensitive and reliable in mutation detection, the occurrence of false negatives cannot be excluded. As an alternative to PCR-SSCP, samples may be analysed for mobility shifts by Denaturing Gradient Gel Electrophoresis (DGGE).6 However, although DGGE appears to be more sensitive than SSCP, it is technically more demanding requiring computer algorithms to assist in the design of appropriate GC-clamped PCR primers. Nucleotide sequencing of suspected mutants observed during DGGE and SSCP is necessary to provide conclusive evidence of mutation. The methodology underlying DNA sequencing, a fundamental molecular genetic technique finding increasing use in molecular pathology, has been revolutionised recently by PCR. Sequencing utilising Tuq DNA polymerase, referred to as cycle sequencing, is carried out in a programmable thermal cycler and is essentially based on the original dideoxynucleotide chain-termination method.’ It involves annealing a radioactively labelled oligonucleotide primer to a single-stranded DNA template and extending the labelled primer in four separate base-specific reactions, each in the presence of higher concentrations of all deoxynucleotides and one chainterminating dideoxynucleotide. The products of these reactions are subsequently separated by electrophoresis on denaturing polyacrylamide gels and visualised by autoradiography. Due to the amplification achieved with the Tuq DNA polymerase, a reduced amount of template is necessary and the high temperatures achieved during the reaction eliminates secondary structure which arises due to intra-strand complementarity in the template strand. Furthermore, the use of Tuq DNA polymerase in cycle sequencing is advantageous in that it yields superior results on double-stranded DNA templates obviating the need to generate asymmetric PCR products or to undertake additional cloning steps prior to sequencing. Automated DNA sequencing offers the opportunity to sequence using fluorescent label (instead of radioactivity) and rapid on-line analysis of data using associated software, facilitates a high through-put of accurate se-
PCR AND MOLECULAR PATHOLOGY
quence. The spectrum of oncogene mutations detectable in human tumours by DNA sequencing may shed some light on disease pathogenesis and on the interaction of various oncoproteins in the transformation of a normal cell to a malignant one. It is likely that direct sequencing by PCR of frequently mutated genes in neoplasia, e.g. ~53, will be combined with immunohistochemical analyses in the correlation of genetic aberrations with changes in protein structure and function and in the determination of the resultant tumour phenotype.
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Detection of polymorphisms and gene loss associated with disease Unrelated individuals may differ in their DNA sequences by as much as one in every 500 bases. These variations or polymorphisms in DNA may either create or destroy a recognition site for a restriction endonuclease, resulting in fragments of differing length after incubation of the DNA fragment with the particular restriction endonuclease. This is termed a Restriction Fragment Length Polymorphism (RFLP). PCR can be used as an alternative to Southern blotting to amplify a region flanking an RFLP and hence to facilitate analysis of the polymorphism. The diagnosis of a wide variety of inherited disorders (e.g. sickle cell anaemia) is based on the detection of RFLPs. If an individual is heterozygous for a polymorphism (i.e. exhibits two distinct alleles, one with and one without the restriction site), the fate of both alleles in disease progression can be monitored. This phenomenon, termed Loss of Heterozygosity (LOH), is particularly useful in the study of neoplasia where tumour suppressor genes are frequently inactivated by allelic deletion (loss of one copy of the gene). Figure 2 shows how allelic deletion at a tumour suppressor gene locus can be determined. A PCR product flanking the known polymorphism is generated from non-tumour and tumour material and digested with the specific restriction endonuclease.8 Fragments corresponding to both alleles (gene copies) can be visualised from an individual heterozygous for the polymorphism. Allelic deletion in the tumour material is detected as absence from the tumour DNA, of a fragment or set of fragments corresponding to an allele. While RFLP-PCR is useful in the study of allelic deletion, at best only 50% of individuals tested will be heterozygous for any particular polymorphism. Tumour DNA only from these heterozygous individuals can be assayed for gene deletion. This is because there are only two possible alleles at any RPLP locus. Microsatellites are another class of polymorphic sequence in DNA which will prove more useful in the analysis of gene deletion in neoplasia. Microsatellites are repeated sequences of the form (CA), which are scattered throughout the genome. The number of CA repeats at any one microsatellite locus can vary from 2 to more than 30. Hence there are multiple possible alleles and the rate of heterozygosity and therefore the informativity in the population increases. Indeed, the average rate of heterozygosity for a microsatellite is 70%.9 The informativity can be increased even further using multiplex-
Fig. P-Detection
of allelic deletion in colorectal tumour. Lanes M shows molecular weight marker (Hae III digested (1x174 DNA), lane 1 shows undigested PCR product flanking the Exon 4 Act II RFLP* in the p53 tumour suppressor gene, lane 2 shows Act II digested PCR product generated from normal peripheral leucocytes of colorectal carcinoma patient; the 259 bp fragment corresponds to one allele and the 160 and 99 bp fragments correspond to the other allele, lane 3 shows digested PCR product from the colorectal tumour; the disappearance of the 259 bp fragment is indicative of Loss of Heterozygosity (LOH) or allelic deletion at the p53 tumour suppressor gene locus.
PCR, incorporating primers for multiple microsatellites in a single PCR. The detection of gene loss in human tumours may provide important prognostic information which may assist in more effective patient management. Allelic deletion at a number of gene loci has been shown to correlate with disease progression in colorectal carcin0ma.l”
Detection of chromosomal translocations by polymerase chain reaction Many diseases including lymphomas and leukaemias exhibit the translocation of one complete chromosome or fragment of a chromosome to another chromosome. Chromosomal translocations contribute to the pathology of a number of diseases because a breakpoint in the gene alters its expression and can result in activation of oncogene expression. For example in SO-90% of follicular lymphomas a translocation between chromosomes 14 and 18, t( 14; 18). occurs resulting from the juxtaposition of the candidate proto-oncogene hrl-2 (B-cell leukaemialymphoma-2) on chromosome 18 with the immunoglobulin heavy chain locus on chromosome 14. At least half of the translocations involving this gene occur within a 150 bp region that is amenable to amplification by PCR.r’ This phenomenon can be detected by PCR amplification of genomic DNA regions flanking the break-point. ” Generation of a PCR product indicates that translocation has occurred and offers support for a diagnosis of follicular lymphoma. In the absence of PCR amplification, a diagnosis cannot be made as alternative translocations may occur. The high sensitivity of the
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PCR affords detection of DNA from 1 in 105 cells, permitting diagnosis under conditions that make the application of morphological or cytogenetic analyses difficult or impossible. Detection of chromosomal translocation by PCR is particularly useful in monitoring remission and relapse in lymphoma patients and is also applicable to retrospective analysis of archival material.12 Detection of pathogens by polymerase chain reaction The detection of both viral and bacterial pathogens represents an important diagnostic area for molecular analysis. The application of PCR to the detection of infectious agents has primarily centred on the diagnosis of viral disease. The tremendous potential of PCR is evident as it eliminates the difficult and often cumbersome process of viral propagation in culture and it also affords increased detection sensitivity relative to conventional techniques. Using PCR it is possible to detect low abundance viral sequences confined to a small subset of cells. Amplification of viral nucleic acids by PCR is a powerful tool for investigating their specific roles in a variety of pathological conditions, and the application of the technique to DNA isolated from paraffin-embedded tissues allows retrospective study of the natural history of infection. The speed with which PCR is accomplished yields rapid diagnosis and provides substantial information facilitating appropriate patient management. To date the nucleotide sequences of several DNA and RNA viruses have been amplified by PCR. DNA viruses including Hepatitis B virus,13 Cytomegalovirus,‘4 Epstein Barr virus,15 Herpes Simplex virus,i6 Human Immunodeficiency virus” and subtypes of Human Papilloma virus18 have been detected in human tissue. Amplification of regions unique to specific subtypes facilitates viral subtype identification. Furthermore, cloning and sequencing of such PCR products potentially yields information regarding mutations and leads to the identification of related viruses. PCR amplification of RNA viral sequences overcomes many problems previously encountered with their detection by conventional techniques. Retroviruses including the Measles virus3 and Enteroviruses19 are detected by reverse transcription of RNA to cDNA and subsequent amplification by PCR. As proviral DNA sequences and viral RNA sequences can be amplified separately, it may be possible to distinguish between latent and proliferative stages of infection using PCR. This would be particularly important in monitoring disease progression. PCR amplification is also applicable to the detection of other pathogens and their typing to the species level. For example, a highly conserved Mycobacterium gene encoding a heat-shock protein can be amplified by PCR. Restriction endonuclease digestion of the resultant PCR product yields fragments of different sizes depending on the Mycobacterium species.20 Detection of gene amplification by polymerase chain reaction Gene amplification is the term used to describe the oc-
currence of multiple copies of a gene within a cell which may result in increased mRNA expression and overabundance of the corresponding protein. The amplification of genes conferring increased growth proliferative potential on a cell, including those for growth factors, growth factor receptors and other oncogenes is commonly encountered in tumour cells. Southern blotting has conventionally been used in the quantitation of gene amplification. However, its ability to accurately quantitate the level of gene amplification is somewhat limited. PCR offers a means to improve the ability to define precise gene copy number by differential PCR. Differential PCR involves the co-amplification (by PCR) of a fragment of a reference gene known to be present as a single copy per haploid genome equivalent and a fragment of the gene being tested; for example, this method has been used in the detection and quantitation of c-erb B2 oncogene amplification in breast turnour.” The technique has been applied to DNA extracted from paraffinembedded material but requires many PCRs to confirm the presence of gene amplification.‘2 Detection of changes in gene expression using polymerase chain reaction Total RNA consists of approximately 80% ribosomal RNA (i-RNA), 15% transfer RNA (tRNA) and 2-5% messenger RNA (mRNA). The n-RNA transcript is formed from the coding DNA sequence and contains the information that defines the subsequently translated protein. Reverse transcription of mRNA using the enzyme Reverse Transcriptase facilitates the synthesis of single stranded complementary DNA copies of the mRNA template (cDNA), which are more stable than RNA and serve as templates for PCR. The reverse transcription reaction may be primed by oligo (dT), (complementary to the mRNA poly A+ tail), random hexamers or gene-specific oligonucleotides. Amplification of the resultant cDNA is accomplished by PCR, generally using gene-specific primers. This technique is referred to as Reverse Transcription-PCR (RT-PCR) and it is applicable to RNA isolated from frozen tissue or archival material. The isolation of RNA free of contaminating genomic DNA is essential for this procedure. An important consideration in designing gene-specific oligonucleotide primers for this technique is that where possible, the primer sequences should not be positioned in the same exon. This ensures that in the event of amplification from contaminating genomic DNA, a product distinguishable in size from the RNA amplification product is generated. The reverse transcription of mRNA species prior to PCR amplification determines whether a particular gene is being expressed and allows quantitation of gene expression levels. Essentially these techniques can be applied to any gene provided sufficient sequence information is available to design appropriate gene-specific primers. Quantitative methods for specific mRNA species using RT-PCR are based on the comparison of the amount of PCR product generated with the amount produced from a known concentration or copy
PCR AND MOLECULAR PATHOLOGY
number of control amplification target in the same reaction. The assessment of gene expression in this manner is especially useful in cases where limited sample is available. Obtaining profiles indicative of gene expression levels in this manner may provide valuable diagnostic insight regarding the development of specific pathological processes. This is of particular importance in neoplasia, where the aberrant expression of a range of proteins, including growth factors, growth factor receptors, intracellular signal transducers or nuclear proteins may occur. Such changes in gene expression levels may have gross effects on cell morphology and function and may confer specific invasive or metastatic properties on the resultant tumour. Recently, a technique has been developed for the identification of differentially expressed mRNA species by a modification of the RTPCR protocol and is termed Differential Display Reverse transcription-PCR (DDRT-PCR).2’-“5 This technique involves reverse transcription of the mRNA primed with oligo (dT) primers ‘anchored’ to the poly A+ tail of the mRNA. This is followed by PCR in the presence of a second IO-mer primer of arbitrary sequence. Theoretically, the use of 20 different arbitrary lo-mer primers enables the visualisation of all expressed genes. The resultant amplified sub-populations of mRNAs are distributed on a DNA sequencing gel. Simultaneous analysis of such fragments from two or more sources identifies differentially expressed genes. The relevant fragments can be excised from the gel and their nucleotide sequence determined. The course of pathological changes occurring in disease may be attributed to single gene mutation or more complex multigene defects which are driven by changes in gene expression. DDRT-PCR has the potential to identify genes pivotal to a broad spectrum of pathological processes and is of immense importance in the analysis of changes in gene expression peculiar to particular cellular events, including differentiation and neoplasia. PCR involving RNA generally requires intact high-quality mRNA species for accurate quantitation of gene expression levels. The inherent poor quality of RNA from archival material renders the technique of limited applicability to the study of paraffinembedded material. However, it is probable that increasing understanding of the clinical significance of altered gene expression, coupled with technical improvements, will result in combined RNA and PCR technologies becoming a routine part of molecular diagnostics.
Establishing polymerase chain reaction in diagnostic molecular pathology Although PCR has vast potential in diagnosis and prognosis. several precautions and optimisation procedures must be undertaken in the routine application of this technique to the analysis of pathological samples. Although a given primer pair may be designed for any of a range of gene-specific target sequences, the conditions under which optimal amplification is achieved vary for each primer pair depending on primer length, composition and product size. It is important that
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no secondary structure exists within the primer sequences and that complementary regions are not present. For each primer the optimal annealing temperature, annealing time and number of PCR cycles must be determined. In addition, the concentration of deoxynucleotide triphosphates and buffer constituents influences the specificity of the reaction and thus optimal concentrations of these components must be determined empirically. In the initial development of a PCR assay it is vital to confirm that the amplification is specific for the desired sequence. This can be achieved by hybridising the product with an oligonucleotide designed to harbour a region of internal sequence. Alternatively a ‘nested’ PCR can be carried out, whereby an additional pair of oligonucleotides are designed for PCR which will amplify a smaller portion of the original PCR product and thus verify its identity. Nested PCR can also be used to increase the specificity of the reaction, particularly in the identification of a highly conserved sequence from related species or it can also be used in cases where a high GC content in the template renders amplification difficult. A ‘hot start’ or heating the reaction mixture prior to the addition of Taq DNA polymerase can also assist in the generation of PCR products from fragments with a high GC content. The inherent sensitivity of PCR which enables amplification from a single target molecule may result in the introduction of an undesired template resulting in artifactual results. To eliminate the possibility of such ‘carry over’ during extraction of DNA from paraffinembedded tissues, it is crucial that the microtome used for sectioning must be clean and excess paraffin and tissue fragments should be removed from the blade with xylene between blocks to minimise the chance of cross contamination of one sample with another. This is of particular importance when samples of both tumour and normal material are being isolated from the same block for analysis. It is also necessary to ensure that cross contamination does not arise due to the inadvertent introduction into fresh samples of previously amplified material (PCR product) as this would also result in artifactual results. False negative results can arise when amplifying nucleic acids extracted from paraffin embedded tissues due to the occasional presence of inhibitors of Tuq DNA polymerase in such samples. The inhibitory factors are effectively removed following extraction with phenolchloroform or by chromatography on Sephadex G-50. It is advisable to designate pre- and post-PCR areas within a laboratory and to maintain the components of the PCR sterile. Positive and negative controls should be carried out with all PCR experiments to exclude the possibility of cross contamination. A positive control may consist of tissue from a culture-proven source of a pathogenic organism or alternatively cloned DNA from an infectious agent also serves as an effective positive control. In negative controls, the template is omitted from the PCR and therefore generation of a product is indicative of contamination.
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One of the latest developments in PCR has been in situ PCR which combines the high sensitivity of DNA amplification by PCR with the anatomical localisation provided by in situ hybridisation. It is particularly advantageous in that particular gene sequences can be localised to individual cells. The methodology involves amplification of specific nucleic acid sequences inside cells, increasing copy numbers to levels detectable by in situ hybridisation or immunohistochemistry. The initial step involves the fixation and permeabilisation of cells or tissue samples to retain morphology and facilitate access of the components of the PCR to the intracellular target sequences. PCR amplification of desired sequences is accomplished either in intact cells as a suspension in eppendorf tubes or directly on cytocentrifuge preparations or tissue sections on glass slides by overlaying the samples with the PCR reaction mixture under a coverslip. Strategies to limit extracellular diffusion of PCR products include overlaying tissue sections with a thin layer of agarosez6 or limitation of PCR cycle number together with the incorporation of biotin-substituted nucleotides into PCR products to render them more bulky.27 Visualisation of amplified intracellular products can be achieved by either in situ hybridisation (indirect in situ PCR) or by direct incorporation of labelled nucleotides (direct in situ hybridisation). In the investigation of viral disease, in situ PCR is invaluable in the identification of cell types harbouring specific viral genetic material, and may allow estimation of viral load in particular cells or tissues. Detection of the DNA viral sequences of Human Immunodeficiency virus,2* Human Papilloma virus29 and Visna virus3o has been achieved. In situ PCR has also been employed in the investigation of endogenous DNA sequences including human single copy genes3’ rearranged cellular genes and chromosomal translocations.32 This technique represents a potentially powerful tool and may assume prime importance in ascertaining the effects of specific therapies. While still emerging as a research technique it is likely that the current rapid technical advances may soon render it applicable to routine diagnosis. Its utility will be evident in situations where the results of immunohistochemistry/PCR/in situ hybridisation may be insufficient to reconcile scientific and diagnostic questions and also in situations where quantitation is important. However, in contrast to other PCR based methods, in situ PCR has had limited success in the analysis of DNA extracted from archival material and its application to archival material awaits additional refinements of the technique.
ability of additional nucleotide sequences for infectious pathogens, genes implicated in cancer and inherited diseases. This will enable the early detection of cancer, the detection of premalignant changes, the detection of potentially metastatic cells in primary tumours and the identification of persons at high risk of disease. It is envisaged that PCR will become routine in pathology laboratories where it will augment diagnostic techniques currently in use. References 1. Saiki R K, Scharf S, Faloona F et al. Enzymatic amplification of P-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science 1985; 230: 135@1354. 2. Shibata D, Martin W J, Amheim N. Analysis of DNA sequences in forty-year-old paraffin-embedded thin-tissue sections: A bridge between molecular biology and classical histology. Cancer Res 1988; 48: 4564-4566. 3. Jackson D P, Quirke P, Lewis F et al. Detection of measles virus RNA in paraffin-embedded tissue. Lancet 1989; i: 1391. 4. Koopmans M, Monroe S S, Coffield L M, Zaki S R. Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives. J Viral Methods 1993; 43: 189-204. 5. Hayashi K. A simple and sensitive method for detection of mutations in the genomic DNA. PCR Methods Applic 1991; 1: 34-38. 6. Myers R M, Fischer S G, Lerman L S, Maniatia T. Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucl Acid Res 1985; 13: 3131-3145. 7. Sanger F, Nicklen S, Coulson A R. DNA sequencing with chain-terminating inhibitors. Proc Nat1 Acad Sci USA 1977; 74: 5463-5467. 8. dela Calle-Martin 0, Fabregat V, Romero M, Soler J, Vives J, Yague J. Act II polymorphism of the ~53 gene. Nucl Acid Res 1990; 18: 4963. 9. Gyapay G, Morissette J, Vignal A et al. The 1993-94 Genethon human genetic linkage map. Nature Genetics 1994; 7: 246-249. IO. Kern S E, Fearon E R, Tersmette K W F et al. Clinical and pathological associations with allelic loss in colorectal carcinomas. J Am Med Assoc 1989; 161: 3099-3103. 11. Bakhshi A, Wright J J, Graninger W et al. Mechanism of the (14, 18) chromosomal translocation: Structural analysis of both derivative 14 and 18 reciprocal partners. Proc Nat1 Acad Sci USA 1987; 84: 23962400. 12. Shibata D, Hu E, Weiss L M, Bymes R K, Nathwani B N. Detection of specific t( 14; 18) chromosomal translocations in fixed tissues. Hum Path01 1990; 21: 199-203. 13. Lampertico P, Malter J S, Colombo M, Gerber M A. Detection of Hepatitis B virus DNA in formalin-fixed, paraffin-embedded tissue by the polymerase chain reaction. Am J Path01 1990; 137: 253-258. 14. Cassol S A, Poon M C, 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-I 11.5. 15. Rouah E, Rogers B B, Wilson D R, Kirkpatrick J B, Buffone G B. Demonstration of Epstein-Barr virus in primary central nervous system lymphomas by the polymerase chain reaction and in situ hvbridisation. Hum Path01 1990: 21: 545-550. . 16. Geradts J, Wamock M, Yen T S B. Use of the polymerase chain reaction in the diagnosis of unsuspected herpes simplex viral oneumonia: Reoort of a case. Hum Path01 1990, 21: 118-121. 17. Shibata D, Byrnes R K, Nathwani B, Kwok S, Sninsky 3, Amheim N. Human Immunodeticiency viral DNA is readily found in lymph node biopsies from seropositive individuals. Analysis of fixed tissues using the polymerase chain reaction. Am J Path01 1989; 135: 697-702. 18. Shibata D K, Amheim N, Martin W J. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J Exp Med 1988; 167: 225-230. 19. Redline R W, Genest D R, Tycko H. Detection of enteroviral infection in paraffin-embedded tissue by the RNA polymerase chain reaction technique. Am J Clin Path01 1991: 96: 568-571. L
Summary It is anticipated that rapidly increasing technical advances in PCR technology will spur the development of additional, equally sensitive techniques. The expanding applications of PCR based methodologies to the study of the molecular pathogenesis of disease rely on the avail-
PCR AND MOLECULAR 20. Telenti A, Marchesi F, Balz M, Bally F, Bottger E C, Bodmer T. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbial 1993; 31: 175-178. 21. Frye R A. Benz C C, Liu E. Detection of amplified oncogenes by differential PCR. Oncogene 1989; 4: 1153-l 157. 22. Neubauer A, Neubauer B, He M et al. Analysis of gene amplification in archival tissues by differential polymerase chain reaction. Oncogene 1992; 7: 1019-1025. 23. Liang P. Pardee A B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 1992; 257: 967-97 1. 24. Liang P, Averboukh L, Pardee A B. Distribution and cloning of eukaryotic messenger RNA by differential display: refinements and optimisation. Nucl Acid Res 1993; 21: 3269-3275. 25. Bauer D, Muller H, Reich .I et al. Identification of differentially expressed mRNA species by an improved display technique (DDRT/PCR). Nucl Acid Res 1993; 21: 4272-4280. 26. Chin K-P, Cohen S H, Morris D W, Jordan G W. Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction. J Histochem Cytochem 1992; 40: 333-341. 27. Komminoth P, Long A A. Ray R. Wolfe H J. In situ polymerase
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chain reaction detection of viral DNA, single copy genes and gene rearrangements in cell suspensions and cytospins. Diagn Mel Path01 1992; I: 85-97. Bagasra 0. Hauptman S P, Lischner H W. Sachs M. Pomerantz R J. Detection of human immunodeficiency virus type I provirus in mononuclear cells by in situ polymerase chain reaction. N Eng J Med 1992; 326: 1385-1391. Nuovo G J. MacConnell P, Forde A, Delvenne P. Detection of human papilloma virus DNA in formalin-fixed tissues by in situ hybridization after amplification by polymerase chain reaction. Am J Pathol 1991; 139: 847-854. Staskus K A, Couch L, Bitterman P et al. In situ amplification of visna virus DNA in tissue sections reveals a reservoir of latently infected cells. Microb Pathol 199 I ; 11: 67-76. Ray R, Komminoth P, Machado M. Wolfe H J. Combined polymerase chain reaction and in situ hybridization for the detection of single copy genes and viral genomic sequences in intact cells. Mod Path01 199 1; 4: 124a. Long A A, Komminoth P, Wolfe H F. Comparison of indirect and direct in-situ polymerase chain reaction in cell preparations and tissue sections. Detection of viral DNA, gene rearrangements and chromosomal translocations. Histochemistry 1993; 99: 151-162.
D. M. Sexton, M. A. Bennett and D. T. Croke
Introduction The aetiology, pathogenesis and prognosis of human disease are routinely assessed by a variety of biochemical and immunohistochemical techniques. However, in recent years an expanding array of molecular biological techniques have increasingly been used as adjuncts to these, and have yielded substantial information contributing to our understanding of the genetic changes which occur during disease progression. Molecular biology is essentially the study of cellular genetic material (DeoxyriboNucleic AcidIRiboNucleic Acid (DNA/RNA)). DNA, tightly packed with proteins, condenses to form chromosomes just before cell division. DNA exists in a highly ordered double helical conformation, consisting of two linear and antiparallel nucleic acid strands. Each strand consists of an alternating sugar-phosphate backbone to which organic bases are attached. The backbone confers structural rigidity on the molecule, while the genetic information is encoded by the sequence of bases. Four possible bases can be incorporated into the DNA molecule, adenine (A), guanine (G), cytosine (C) and thymine (T). Base pairing between the two antiparallel strands follows a set pattern in which A always pairs with T and C always pairs with G. This is referred to as complementarity. A sequence of DNA which encodes a single protein is referred to as a gene. Structurally, eucaryotic genes consist of regions coding for protein, called exons, which are separated by noncoding intervening sequences referred to as introns. DNA in the cell of any one human individual contains information for about 100 000 genes, only about 15% of which are expressed in a specific cell type, conferring defined structural and functional
D. M. Sexton, M. A. Bennett and D. T. Croke, Department of Biochemistry, Royal College of Surgeons in Ireland, 123, St Stephens Green, Dublin 2, Ireland
properties on the cell. An expressed protein is transcribed from DNA to an intermediate known as messenger RNA (mRNA). Base triplets (codons) determine the incorporation of amino acids into the newly synthesised protein in the order determined by the original DNA strand. To date the nucleotide sequences of approximately 3000 human genes are known. Rapid progress in the identification and characterisation of gene sequences is being made by the Human Genome Project which hopes to generate a physical map of the entire human genome by the end of this decade. Genetic changes underlie the development of many diseases due to aberrant gene expression. As the nucleotide sequences of genes become known, the elucidation of specific diseasecausing genetic aberrations including point mutations, deletions (loss of gene sequence), or translocations will contribute immensely to our understanding of the pathological characteristics of disease. Pathogenic mutations may occur in exons, introns or regulatory regions of genes. The contribution of a pathological mutation in a single gene to the clinical phenotype is dependent on the dominance/recessivity of the mutant gene in relation to the normal gene, the proportion of mature cells in which the mutant gene is present and in some cases the parental origin of the mutation.
The polymerase chain reaction The most exciting and innovative molecular biology technique developed to date is the PoIymerase Chain Reaction (PCR) which offers immense promise in diagnostic and prognostic applications. PCR represents a powerful in vitro method which, due to its sensitivity, speed and selectivity has superseded previous conventional molecular biological techniques, such as Southern blotting analysis and in situ hybridisation, in biomedical research. The method essentially involves the selective amplification of specific segments of DNA.’ The basic
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aquaticus renders the process amenable to automation. Amplification of RNA is also possible but requires a reverse transcription step prior to PCR. The quality and quantity of target nucleic acid required for PCR analysis represent features of the technique that are very attractive to the pathologist. In contrast to conventional molecular biological techniques, much smaller quantities of DNA are required to amplify a specific sequence by PCR. Nanogram or even picogram concentrations of template are sufficient for PCR; conventional molecular biological techniques require micrograms of nucleic acid. High quality DNA can be isolated from fresh or frozen tissue. However, many pathology laboratories possess a vast repertoire of human tissues fixed
reaction, carried out in a programmable thermocycler, involves repetitive cycles of DNA synthesis. Each cycle consists of three steps (Fig. 1). The first step involves denaturation of the target nucleic acid which renders it single stranded. This is followed by annealing of synthetic oligonucleotide primers specifically designed to hybridise to the target nucleic acid region. The third step involves extension from the annealed primers, catalysed by a DNA polymerase enzyme. In a typical PCR analysis 20-40 such cycles are carried out. As successive products are generated these become templates for subsequent cycles resulting in exponential amplification of the target region. The use of the thermostable Taq DNA polymerase from the thermophilic bacterium Thermus Double-stranded
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Fig. l-Diagrammatic representation of the three steps involved in a PCR cycle. Step 1 involves denaturation of the target nucleic acid tb its single stranded form (typically 94°C). In step 2 annealing takes place: oligonucleotide primers hybridise to their complementary regions on the target nucleic acid sequence (37-65°C). In step 3 there is extension of the annealed primers using Taq DNA polymerase (72°C). Repeated cycles of PCR amplification, using a programmable thermocycler, results in a exponential increase in the copy - number of sequence.
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in formalin and embedded in paraffin. Fixation with formalin causes cross-linking between DNA and proteins and results in the DNA becoming more rigid and susceptible to mechanical shearing during extraction and consequently can have a profound effect on the integrity of extracted DNA. Several methods, generally involving proteinase K digestion, have been reported for the extraction of DNA from paraffin-embedded tissue. As PCR does not require a high molecular weight template, the crude and somewhat degraded DNA extracted from paraffin-embedded tissue is amenable to amplification by PCR and fragments of up to 500 base pairs are attainable. The potential to use DNA extracted from archival material facilitates retrospective studies on large numbers of patients, making it possible to chronologically trace the molecular progression of disease. A single 5 urn section from formalin-fixed paraffin-embedded tissue contains enough DNA even after 40 years to serve as a template in PCR.2 The labile nature of RNA and its susceptibility to degradation by ubiquitous ribonuclease enzymes renders its isolation inherently more difficult than that of DNA. A procedure for the isolation of RNA from paraflinembedded tissue based on the standard DNA extraction protocol has been reported.3 More recently a rapid method based on the binding of RNA to acid-treated glass beads in the presence of a high concentration of guanidinium was described.“ RNA amplification products of up to 500 base pairs have been obtained from RNA isolated by this procedure. The efficient isolation of RNA from archival material has immense implications in diagnostic pathology as it provides a means to understand changes in gene expression occurring during disease pathogenesis. The innovative yet simple basic PCR results in the generation of millions of copies of a particular target fragment, which can be used directly in diagnosis or can be subjected to further analysis to reveal detailed information regarding genetic mechanisms in disease pathogenesis. A number of modifications of the original PCR have been developed which allow the identification and characterisation of viral and microbial pathogens, detection of point mutations underlying a range of genetic diseases and analysis of changes in gene expression and of gene rearrangements peculiar to specific pathological processes. The following techniques represent some of the variations of PCR applicable to the investigation of disease, and will enable pathologists to evaluate resected tissues and archival material for a range of genetic abnormalities. Detection of disease causing mutations using polymerase chain reaction Detection of point-mutations underlying genetic disease by PCR technology can be accomplished by a sensitive method termed PCR-Single Strand Conformation Polymorphism (PCR-SSCP) which is based on the ability of a single point-mutation to alter the conformation and consequent migration of single-stranded DNA during
electrophoresis. In PCR-SSCP the target sequence in genomic DNA or cDNA is simultaneously amplified and labelled by the incorporation of radioactive primers or nucleotides which allow subsequent detection of the DNA fragment by autoradiography. Following amplification, the double-stranded PCR product is denatured to its single-stranded form and subjected to non-denaturing gel electrophoresis. The conformation (folding pattern) of the single-stranded DNA, which is dictated by the nucleotide sequence, determines its electrophoretic mobility. Assuming that all base changes alter conformation, the presence of a mutation is reflected in a comparative mobility shift between a mutant and a wildtype sequence. The sensitivity of PCR-SSCP in detecting point mutations, which can reach up to 1OO%,5is dependent on several factors including fragment length, gel constituents and temperature of electrophoresis. This technique facilitates screening for activating mutations in oncogenes, inactivating mutations in tumour suppressor genes and underlying mutations in inherited disorders. While this technique is sensitive and reliable in mutation detection, the occurrence of false negatives cannot be excluded. As an alternative to PCR-SSCP, samples may be analysed for mobility shifts by Denaturing Gradient Gel Electrophoresis (DGGE).6 However, although DGGE appears to be more sensitive than SSCP, it is technically more demanding requiring computer algorithms to assist in the design of appropriate GC-clamped PCR primers. Nucleotide sequencing of suspected mutants observed during DGGE and SSCP is necessary to provide conclusive evidence of mutation. The methodology underlying DNA sequencing, a fundamental molecular genetic technique finding increasing use in molecular pathology, has been revolutionised recently by PCR. Sequencing utilising Tuq DNA polymerase, referred to as cycle sequencing, is carried out in a programmable thermal cycler and is essentially based on the original dideoxynucleotide chain-termination method.’ It involves annealing a radioactively labelled oligonucleotide primer to a single-stranded DNA template and extending the labelled primer in four separate base-specific reactions, each in the presence of higher concentrations of all deoxynucleotides and one chainterminating dideoxynucleotide. The products of these reactions are subsequently separated by electrophoresis on denaturing polyacrylamide gels and visualised by autoradiography. Due to the amplification achieved with the Tuq DNA polymerase, a reduced amount of template is necessary and the high temperatures achieved during the reaction eliminates secondary structure which arises due to intra-strand complementarity in the template strand. Furthermore, the use of Tuq DNA polymerase in cycle sequencing is advantageous in that it yields superior results on double-stranded DNA templates obviating the need to generate asymmetric PCR products or to undertake additional cloning steps prior to sequencing. Automated DNA sequencing offers the opportunity to sequence using fluorescent label (instead of radioactivity) and rapid on-line analysis of data using associated software, facilitates a high through-put of accurate se-
PCR AND MOLECULAR PATHOLOGY
quence. The spectrum of oncogene mutations detectable in human tumours by DNA sequencing may shed some light on disease pathogenesis and on the interaction of various oncoproteins in the transformation of a normal cell to a malignant one. It is likely that direct sequencing by PCR of frequently mutated genes in neoplasia, e.g. ~53, will be combined with immunohistochemical analyses in the correlation of genetic aberrations with changes in protein structure and function and in the determination of the resultant tumour phenotype.
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Detection of polymorphisms and gene loss associated with disease Unrelated individuals may differ in their DNA sequences by as much as one in every 500 bases. These variations or polymorphisms in DNA may either create or destroy a recognition site for a restriction endonuclease, resulting in fragments of differing length after incubation of the DNA fragment with the particular restriction endonuclease. This is termed a Restriction Fragment Length Polymorphism (RFLP). PCR can be used as an alternative to Southern blotting to amplify a region flanking an RFLP and hence to facilitate analysis of the polymorphism. The diagnosis of a wide variety of inherited disorders (e.g. sickle cell anaemia) is based on the detection of RFLPs. If an individual is heterozygous for a polymorphism (i.e. exhibits two distinct alleles, one with and one without the restriction site), the fate of both alleles in disease progression can be monitored. This phenomenon, termed Loss of Heterozygosity (LOH), is particularly useful in the study of neoplasia where tumour suppressor genes are frequently inactivated by allelic deletion (loss of one copy of the gene). Figure 2 shows how allelic deletion at a tumour suppressor gene locus can be determined. A PCR product flanking the known polymorphism is generated from non-tumour and tumour material and digested with the specific restriction endonuclease.8 Fragments corresponding to both alleles (gene copies) can be visualised from an individual heterozygous for the polymorphism. Allelic deletion in the tumour material is detected as absence from the tumour DNA, of a fragment or set of fragments corresponding to an allele. While RFLP-PCR is useful in the study of allelic deletion, at best only 50% of individuals tested will be heterozygous for any particular polymorphism. Tumour DNA only from these heterozygous individuals can be assayed for gene deletion. This is because there are only two possible alleles at any RPLP locus. Microsatellites are another class of polymorphic sequence in DNA which will prove more useful in the analysis of gene deletion in neoplasia. Microsatellites are repeated sequences of the form (CA), which are scattered throughout the genome. The number of CA repeats at any one microsatellite locus can vary from 2 to more than 30. Hence there are multiple possible alleles and the rate of heterozygosity and therefore the informativity in the population increases. Indeed, the average rate of heterozygosity for a microsatellite is 70%.9 The informativity can be increased even further using multiplex-
Fig. P-Detection
of allelic deletion in colorectal tumour. Lanes M shows molecular weight marker (Hae III digested (1x174 DNA), lane 1 shows undigested PCR product flanking the Exon 4 Act II RFLP* in the p53 tumour suppressor gene, lane 2 shows Act II digested PCR product generated from normal peripheral leucocytes of colorectal carcinoma patient; the 259 bp fragment corresponds to one allele and the 160 and 99 bp fragments correspond to the other allele, lane 3 shows digested PCR product from the colorectal tumour; the disappearance of the 259 bp fragment is indicative of Loss of Heterozygosity (LOH) or allelic deletion at the p53 tumour suppressor gene locus.
PCR, incorporating primers for multiple microsatellites in a single PCR. The detection of gene loss in human tumours may provide important prognostic information which may assist in more effective patient management. Allelic deletion at a number of gene loci has been shown to correlate with disease progression in colorectal carcin0ma.l”
Detection of chromosomal translocations by polymerase chain reaction Many diseases including lymphomas and leukaemias exhibit the translocation of one complete chromosome or fragment of a chromosome to another chromosome. Chromosomal translocations contribute to the pathology of a number of diseases because a breakpoint in the gene alters its expression and can result in activation of oncogene expression. For example in SO-90% of follicular lymphomas a translocation between chromosomes 14 and 18, t( 14; 18). occurs resulting from the juxtaposition of the candidate proto-oncogene hrl-2 (B-cell leukaemialymphoma-2) on chromosome 18 with the immunoglobulin heavy chain locus on chromosome 14. At least half of the translocations involving this gene occur within a 150 bp region that is amenable to amplification by PCR.r’ This phenomenon can be detected by PCR amplification of genomic DNA regions flanking the break-point. ” Generation of a PCR product indicates that translocation has occurred and offers support for a diagnosis of follicular lymphoma. In the absence of PCR amplification, a diagnosis cannot be made as alternative translocations may occur. The high sensitivity of the
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PCR affords detection of DNA from 1 in 105 cells, permitting diagnosis under conditions that make the application of morphological or cytogenetic analyses difficult or impossible. Detection of chromosomal translocation by PCR is particularly useful in monitoring remission and relapse in lymphoma patients and is also applicable to retrospective analysis of archival material.12 Detection of pathogens by polymerase chain reaction The detection of both viral and bacterial pathogens represents an important diagnostic area for molecular analysis. The application of PCR to the detection of infectious agents has primarily centred on the diagnosis of viral disease. The tremendous potential of PCR is evident as it eliminates the difficult and often cumbersome process of viral propagation in culture and it also affords increased detection sensitivity relative to conventional techniques. Using PCR it is possible to detect low abundance viral sequences confined to a small subset of cells. Amplification of viral nucleic acids by PCR is a powerful tool for investigating their specific roles in a variety of pathological conditions, and the application of the technique to DNA isolated from paraffin-embedded tissues allows retrospective study of the natural history of infection. The speed with which PCR is accomplished yields rapid diagnosis and provides substantial information facilitating appropriate patient management. To date the nucleotide sequences of several DNA and RNA viruses have been amplified by PCR. DNA viruses including Hepatitis B virus,13 Cytomegalovirus,‘4 Epstein Barr virus,15 Herpes Simplex virus,i6 Human Immunodeficiency virus” and subtypes of Human Papilloma virus18 have been detected in human tissue. Amplification of regions unique to specific subtypes facilitates viral subtype identification. Furthermore, cloning and sequencing of such PCR products potentially yields information regarding mutations and leads to the identification of related viruses. PCR amplification of RNA viral sequences overcomes many problems previously encountered with their detection by conventional techniques. Retroviruses including the Measles virus3 and Enteroviruses19 are detected by reverse transcription of RNA to cDNA and subsequent amplification by PCR. As proviral DNA sequences and viral RNA sequences can be amplified separately, it may be possible to distinguish between latent and proliferative stages of infection using PCR. This would be particularly important in monitoring disease progression. PCR amplification is also applicable to the detection of other pathogens and their typing to the species level. For example, a highly conserved Mycobacterium gene encoding a heat-shock protein can be amplified by PCR. Restriction endonuclease digestion of the resultant PCR product yields fragments of different sizes depending on the Mycobacterium species.20 Detection of gene amplification by polymerase chain reaction Gene amplification is the term used to describe the oc-
currence of multiple copies of a gene within a cell which may result in increased mRNA expression and overabundance of the corresponding protein. The amplification of genes conferring increased growth proliferative potential on a cell, including those for growth factors, growth factor receptors and other oncogenes is commonly encountered in tumour cells. Southern blotting has conventionally been used in the quantitation of gene amplification. However, its ability to accurately quantitate the level of gene amplification is somewhat limited. PCR offers a means to improve the ability to define precise gene copy number by differential PCR. Differential PCR involves the co-amplification (by PCR) of a fragment of a reference gene known to be present as a single copy per haploid genome equivalent and a fragment of the gene being tested; for example, this method has been used in the detection and quantitation of c-erb B2 oncogene amplification in breast turnour.” The technique has been applied to DNA extracted from paraffinembedded material but requires many PCRs to confirm the presence of gene amplification.‘2 Detection of changes in gene expression using polymerase chain reaction Total RNA consists of approximately 80% ribosomal RNA (i-RNA), 15% transfer RNA (tRNA) and 2-5% messenger RNA (mRNA). The n-RNA transcript is formed from the coding DNA sequence and contains the information that defines the subsequently translated protein. Reverse transcription of mRNA using the enzyme Reverse Transcriptase facilitates the synthesis of single stranded complementary DNA copies of the mRNA template (cDNA), which are more stable than RNA and serve as templates for PCR. The reverse transcription reaction may be primed by oligo (dT), (complementary to the mRNA poly A+ tail), random hexamers or gene-specific oligonucleotides. Amplification of the resultant cDNA is accomplished by PCR, generally using gene-specific primers. This technique is referred to as Reverse Transcription-PCR (RT-PCR) and it is applicable to RNA isolated from frozen tissue or archival material. The isolation of RNA free of contaminating genomic DNA is essential for this procedure. An important consideration in designing gene-specific oligonucleotide primers for this technique is that where possible, the primer sequences should not be positioned in the same exon. This ensures that in the event of amplification from contaminating genomic DNA, a product distinguishable in size from the RNA amplification product is generated. The reverse transcription of mRNA species prior to PCR amplification determines whether a particular gene is being expressed and allows quantitation of gene expression levels. Essentially these techniques can be applied to any gene provided sufficient sequence information is available to design appropriate gene-specific primers. Quantitative methods for specific mRNA species using RT-PCR are based on the comparison of the amount of PCR product generated with the amount produced from a known concentration or copy
PCR AND MOLECULAR PATHOLOGY
number of control amplification target in the same reaction. The assessment of gene expression in this manner is especially useful in cases where limited sample is available. Obtaining profiles indicative of gene expression levels in this manner may provide valuable diagnostic insight regarding the development of specific pathological processes. This is of particular importance in neoplasia, where the aberrant expression of a range of proteins, including growth factors, growth factor receptors, intracellular signal transducers or nuclear proteins may occur. Such changes in gene expression levels may have gross effects on cell morphology and function and may confer specific invasive or metastatic properties on the resultant tumour. Recently, a technique has been developed for the identification of differentially expressed mRNA species by a modification of the RTPCR protocol and is termed Differential Display Reverse transcription-PCR (DDRT-PCR).2’-“5 This technique involves reverse transcription of the mRNA primed with oligo (dT) primers ‘anchored’ to the poly A+ tail of the mRNA. This is followed by PCR in the presence of a second IO-mer primer of arbitrary sequence. Theoretically, the use of 20 different arbitrary lo-mer primers enables the visualisation of all expressed genes. The resultant amplified sub-populations of mRNAs are distributed on a DNA sequencing gel. Simultaneous analysis of such fragments from two or more sources identifies differentially expressed genes. The relevant fragments can be excised from the gel and their nucleotide sequence determined. The course of pathological changes occurring in disease may be attributed to single gene mutation or more complex multigene defects which are driven by changes in gene expression. DDRT-PCR has the potential to identify genes pivotal to a broad spectrum of pathological processes and is of immense importance in the analysis of changes in gene expression peculiar to particular cellular events, including differentiation and neoplasia. PCR involving RNA generally requires intact high-quality mRNA species for accurate quantitation of gene expression levels. The inherent poor quality of RNA from archival material renders the technique of limited applicability to the study of paraffinembedded material. However, it is probable that increasing understanding of the clinical significance of altered gene expression, coupled with technical improvements, will result in combined RNA and PCR technologies becoming a routine part of molecular diagnostics.
Establishing polymerase chain reaction in diagnostic molecular pathology Although PCR has vast potential in diagnosis and prognosis. several precautions and optimisation procedures must be undertaken in the routine application of this technique to the analysis of pathological samples. Although a given primer pair may be designed for any of a range of gene-specific target sequences, the conditions under which optimal amplification is achieved vary for each primer pair depending on primer length, composition and product size. It is important that
23 1
no secondary structure exists within the primer sequences and that complementary regions are not present. For each primer the optimal annealing temperature, annealing time and number of PCR cycles must be determined. In addition, the concentration of deoxynucleotide triphosphates and buffer constituents influences the specificity of the reaction and thus optimal concentrations of these components must be determined empirically. In the initial development of a PCR assay it is vital to confirm that the amplification is specific for the desired sequence. This can be achieved by hybridising the product with an oligonucleotide designed to harbour a region of internal sequence. Alternatively a ‘nested’ PCR can be carried out, whereby an additional pair of oligonucleotides are designed for PCR which will amplify a smaller portion of the original PCR product and thus verify its identity. Nested PCR can also be used to increase the specificity of the reaction, particularly in the identification of a highly conserved sequence from related species or it can also be used in cases where a high GC content in the template renders amplification difficult. A ‘hot start’ or heating the reaction mixture prior to the addition of Taq DNA polymerase can also assist in the generation of PCR products from fragments with a high GC content. The inherent sensitivity of PCR which enables amplification from a single target molecule may result in the introduction of an undesired template resulting in artifactual results. To eliminate the possibility of such ‘carry over’ during extraction of DNA from paraffinembedded tissues, it is crucial that the microtome used for sectioning must be clean and excess paraffin and tissue fragments should be removed from the blade with xylene between blocks to minimise the chance of cross contamination of one sample with another. This is of particular importance when samples of both tumour and normal material are being isolated from the same block for analysis. It is also necessary to ensure that cross contamination does not arise due to the inadvertent introduction into fresh samples of previously amplified material (PCR product) as this would also result in artifactual results. False negative results can arise when amplifying nucleic acids extracted from paraffin embedded tissues due to the occasional presence of inhibitors of Tuq DNA polymerase in such samples. The inhibitory factors are effectively removed following extraction with phenolchloroform or by chromatography on Sephadex G-50. It is advisable to designate pre- and post-PCR areas within a laboratory and to maintain the components of the PCR sterile. Positive and negative controls should be carried out with all PCR experiments to exclude the possibility of cross contamination. A positive control may consist of tissue from a culture-proven source of a pathogenic organism or alternatively cloned DNA from an infectious agent also serves as an effective positive control. In negative controls, the template is omitted from the PCR and therefore generation of a product is indicative of contamination.
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One of the latest developments in PCR has been in situ PCR which combines the high sensitivity of DNA amplification by PCR with the anatomical localisation provided by in situ hybridisation. It is particularly advantageous in that particular gene sequences can be localised to individual cells. The methodology involves amplification of specific nucleic acid sequences inside cells, increasing copy numbers to levels detectable by in situ hybridisation or immunohistochemistry. The initial step involves the fixation and permeabilisation of cells or tissue samples to retain morphology and facilitate access of the components of the PCR to the intracellular target sequences. PCR amplification of desired sequences is accomplished either in intact cells as a suspension in eppendorf tubes or directly on cytocentrifuge preparations or tissue sections on glass slides by overlaying the samples with the PCR reaction mixture under a coverslip. Strategies to limit extracellular diffusion of PCR products include overlaying tissue sections with a thin layer of agarosez6 or limitation of PCR cycle number together with the incorporation of biotin-substituted nucleotides into PCR products to render them more bulky.27 Visualisation of amplified intracellular products can be achieved by either in situ hybridisation (indirect in situ PCR) or by direct incorporation of labelled nucleotides (direct in situ hybridisation). In the investigation of viral disease, in situ PCR is invaluable in the identification of cell types harbouring specific viral genetic material, and may allow estimation of viral load in particular cells or tissues. Detection of the DNA viral sequences of Human Immunodeficiency virus,2* Human Papilloma virus29 and Visna virus3o has been achieved. In situ PCR has also been employed in the investigation of endogenous DNA sequences including human single copy genes3’ rearranged cellular genes and chromosomal translocations.32 This technique represents a potentially powerful tool and may assume prime importance in ascertaining the effects of specific therapies. While still emerging as a research technique it is likely that the current rapid technical advances may soon render it applicable to routine diagnosis. Its utility will be evident in situations where the results of immunohistochemistry/PCR/in situ hybridisation may be insufficient to reconcile scientific and diagnostic questions and also in situations where quantitation is important. However, in contrast to other PCR based methods, in situ PCR has had limited success in the analysis of DNA extracted from archival material and its application to archival material awaits additional refinements of the technique.
ability of additional nucleotide sequences for infectious pathogens, genes implicated in cancer and inherited diseases. This will enable the early detection of cancer, the detection of premalignant changes, the detection of potentially metastatic cells in primary tumours and the identification of persons at high risk of disease. It is envisaged that PCR will become routine in pathology laboratories where it will augment diagnostic techniques currently in use. References 1. Saiki R K, Scharf S, Faloona F et al. Enzymatic amplification of P-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science 1985; 230: 135@1354. 2. Shibata D, Martin W J, Amheim N. Analysis of DNA sequences in forty-year-old paraffin-embedded thin-tissue sections: A bridge between molecular biology and classical histology. Cancer Res 1988; 48: 4564-4566. 3. Jackson D P, Quirke P, Lewis F et al. Detection of measles virus RNA in paraffin-embedded tissue. Lancet 1989; i: 1391. 4. Koopmans M, Monroe S S, Coffield L M, Zaki S R. Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives. J Viral Methods 1993; 43: 189-204. 5. Hayashi K. A simple and sensitive method for detection of mutations in the genomic DNA. PCR Methods Applic 1991; 1: 34-38. 6. Myers R M, Fischer S G, Lerman L S, Maniatia T. Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucl Acid Res 1985; 13: 3131-3145. 7. Sanger F, Nicklen S, Coulson A R. DNA sequencing with chain-terminating inhibitors. Proc Nat1 Acad Sci USA 1977; 74: 5463-5467. 8. dela Calle-Martin 0, Fabregat V, Romero M, Soler J, Vives J, Yague J. Act II polymorphism of the ~53 gene. Nucl Acid Res 1990; 18: 4963. 9. Gyapay G, Morissette J, Vignal A et al. The 1993-94 Genethon human genetic linkage map. Nature Genetics 1994; 7: 246-249. IO. Kern S E, Fearon E R, Tersmette K W F et al. Clinical and pathological associations with allelic loss in colorectal carcinomas. J Am Med Assoc 1989; 161: 3099-3103. 11. Bakhshi A, Wright J J, Graninger W et al. Mechanism of the (14, 18) chromosomal translocation: Structural analysis of both derivative 14 and 18 reciprocal partners. Proc Nat1 Acad Sci USA 1987; 84: 23962400. 12. Shibata D, Hu E, Weiss L M, Bymes R K, Nathwani B N. Detection of specific t( 14; 18) chromosomal translocations in fixed tissues. Hum Path01 1990; 21: 199-203. 13. Lampertico P, Malter J S, Colombo M, Gerber M A. Detection of Hepatitis B virus DNA in formalin-fixed, paraffin-embedded tissue by the polymerase chain reaction. Am J Path01 1990; 137: 253-258. 14. Cassol S A, Poon M C, 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-I 11.5. 15. Rouah E, Rogers B B, Wilson D R, Kirkpatrick J B, Buffone G B. Demonstration of Epstein-Barr virus in primary central nervous system lymphomas by the polymerase chain reaction and in situ hvbridisation. Hum Path01 1990: 21: 545-550. . 16. Geradts J, Wamock M, Yen T S B. Use of the polymerase chain reaction in the diagnosis of unsuspected herpes simplex viral oneumonia: Reoort of a case. Hum Path01 1990, 21: 118-121. 17. Shibata D, Byrnes R K, Nathwani B, Kwok S, Sninsky 3, Amheim N. Human Immunodeticiency viral DNA is readily found in lymph node biopsies from seropositive individuals. Analysis of fixed tissues using the polymerase chain reaction. Am J Path01 1989; 135: 697-702. 18. Shibata D K, Amheim N, Martin W J. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J Exp Med 1988; 167: 225-230. 19. Redline R W, Genest D R, Tycko H. Detection of enteroviral infection in paraffin-embedded tissue by the RNA polymerase chain reaction technique. Am J Clin Path01 1991: 96: 568-571. L
Summary It is anticipated that rapidly increasing technical advances in PCR technology will spur the development of additional, equally sensitive techniques. The expanding applications of PCR based methodologies to the study of the molecular pathogenesis of disease rely on the avail-
PCR AND MOLECULAR 20. Telenti A, Marchesi F, Balz M, Bally F, Bottger E C, Bodmer T. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbial 1993; 31: 175-178. 21. Frye R A. Benz C C, Liu E. Detection of amplified oncogenes by differential PCR. Oncogene 1989; 4: 1153-l 157. 22. Neubauer A, Neubauer B, He M et al. Analysis of gene amplification in archival tissues by differential polymerase chain reaction. Oncogene 1992; 7: 1019-1025. 23. Liang P. Pardee A B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 1992; 257: 967-97 1. 24. Liang P, Averboukh L, Pardee A B. Distribution and cloning of eukaryotic messenger RNA by differential display: refinements and optimisation. Nucl Acid Res 1993; 21: 3269-3275. 25. Bauer D, Muller H, Reich .I et al. Identification of differentially expressed mRNA species by an improved display technique (DDRT/PCR). Nucl Acid Res 1993; 21: 4272-4280. 26. Chin K-P, Cohen S H, Morris D W, Jordan G W. Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction. J Histochem Cytochem 1992; 40: 333-341. 27. Komminoth P, Long A A. Ray R. Wolfe H J. In situ polymerase
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