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In Situ PCR A Review

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In Situ PCR A Review

[Frontiers in Bioscience 2, c15-29, September 1, 1997]

    IN SITU PCR. OVERVIEW OF PROCEDURES AND APPLICATIONS

     Carlos A. Muro-Cacho, M.D., Ph.D.

Department of Pathology, H. Lee Moffitt Cancer Center and Research Institute and University of South Florida College of

    Medicine, Tampa, Florida, USA

TABLE OF CONTENTS

     1. Abstract

     2. Introduction

     3. General considerations

     3.1. Equipment

     3.2 Technical aspects

     3.3 Types of samples

     3.4 Fixation

     3.5 Proteolytic digestion

     3.6 Additional pretreatments

     4. Polymerase Chain Reaction (PCR)

    4.1 Polymerases

    4.2 Deoxynucleotide triphosphates (dNTPs)

    4.3 Buffers

    4.4 Primers

    4.5 Cycling profile

     5. In situ PCR Protocols

    5.1 In situ RT-PCR

    5.2 Direct In situ PCR

    5.3 Indirect In situ PCR

     6. Controls

    6.1 Causes of false negativity

    6.2 Causes of false positivity

    6.3 DNA repair

    6.4 Mispriming

    6.5 Endogenous priming

    6.6 Diffusion of PCR products

     7. Applications

     8. Acknowledgment

     9. References

     10. Appendices

     Appendix 1 - General information

     Appendix 2 - Sample preparation Appendix 3 - Proteolytic digestion Appendix 4 - Additional pretreatments Appendix 5 - RT-PCR Appendix 6 - Polymerase Chain Reaction Appendix 7 - Direct In situ PCR Appendix 8 - Indirect In situ PCR Appendix 9 - Detection methods morphology is essential to the full understanding of cell 1. ABSTRACT biology. A variety of methods for detection of nucleic acids are currently available. Solution PCR requires disruption of The evaluation of gene expression in the context of cellular the sample and detection of the amplified material by _____________________________________________________ electrophoresis in agarose gels. In situ Hybridization Received 1/10/97 Accepted 7/15/97 methods, on the other hand, permit morphological correlation Send correspondence to: Carlos A. Muro-Cacho, M.D., Ph.D. and provide a high sensitivity that is sufficient for many Pathology Department, H.Lee Moffitt Cancer Center and applications. In some instances, however, the amount of Research Institute at University of South Florida, College of target in the sample is below the limit of detection of this Medicine, 12902 Magnolia Drive, Tampa, FL 33612-9497 technique. In situ PCR allows the detection of minimal Tel: (813) 972-4673 ext. 2268, FAX:(813) 632-1708, E-mail: amounts of nucleic acids with exquisite sensitivity and murocacho@moffitt.usf.edu

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In Situ PCR; An Overview

    specificity, while the integrity of the cells and the morphology procedures that would be otherwise restricted to highly of tissues remains preserved. This technique, although not specialized basic science research groups. The adaptation of exempt from difficulties, is undergoing methodological some of these procedures, to the detection of nucleic acids in simplifications that will make it suitable for an increasing cells and tissues, converts genetic information into a visual number of basic science and clinical applications. The signal that can be evaluated In situ while preserving cellular

    following is a review of the principles, methods and integrity and tissue morphology (1-16). This signal can then applications of In situ PCR. be evaluated in the context of a homogeneous or

     heterogeneous cell population, a given cellular compartment, 2. INTRODUCTION or a particular pathological tissue change. In situ PCR is also

     known as PCR In situ (1), PCR ISH (2), In-cell PCR (3), and

    The last decade has witnessed a continuous PCR-driven ISH (4), and for those modifications intended to revolution in molecular biology methods and the parallel amplify mRNA, RT In situ PCR (5) or In situ cDNA PCR (6) development of a competitive biotechnology industry that has (appendix 1 and figure 1). made available high quality reagents and sophisticated equipments at a progressively lower cost. This has allowed the routine use, by many laboratories, of state-of-the-art

     SAMPLE PREPARATION

     FIXATION

     PARAFFIN EMBEDDING

     SECTIONING AND SLIDE PREPARATION

     DEPARAFFINATION

     REHYDRATION

     PROTEOLYTIC DIGESTION

     IN SITU PCR IN SITU RT-PCR

     DNase DIGESTION (optional)

     RT

     DNA AMPLIFICATION

     DIRECT INDIRECT

     IHC ISH

     VISUALIZATION

    Figure 1. In situ PCR protocols for tissue sections

Abbreviations: PCR = Polymerase Chain Reaction, RT = Reverse Transcription, ISH = In situ Hybridization, IHC =

    Immunohistochemistry

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In Situ PCR; An Overview

    polish application and removal, transfers in and out of the 3. GENERAL CONSIDERATIONS

    thermocycler, etc.).

     3.1 Equipment

     Thermocyclers are designed to reach and maintain 3.3 Types of samples (appendix 2)

    the necessary temperatures in a reproducible manner over In situ PCR has been successfully performed on a specified periods of time and predetermined numbers of variety of samples: cell smears, imprints, cell blocks, cycles. Some of these instruments have independent "slide" cytospins, metaphase chromosomes, frozen sections and blocks and "tube" blocks, connected to a common sections of tissues embedded in plastic or paraffin. Haase et programmable controlling unit, providing the opportunity to al (14) performed In situ PCR on fixed cells suspended in the run different protocols simultaneously and to correlate PCR reaction mixture within a microcentrifuge tube. In this solution-PCR with In situ PCR results. The commercially procedure, after amplification, cells are recovered by available thermocyclers, use different mechanisms to prevent cytocentrifugation onto glass slides (3, 4, 8, 12, 14). An evaporation and increase efficiency and consistency of results. advantage of this method is that cells may be lysed after In the Omnislide Temperature Cycling System, Hybaid amplification and the lysate analyzed by gel electrophoresis (Teddington, Middlesex, UK, http://www.hybaid.co.uk/), and Southern blot hybridization (14, 15). Good results can slides are placed in a sealed humid chamber. In the PTC-also be obtained in cytospins and cell blocks prepared 100-12MS Programmable Thermal Controller (MJ Research, following standard protocols. When tissues are used, 4-6 Inc., Watertown, MA, USA, http://www.mjr.com/), the micron thick sections are prepared from paraffin or frozen reagents are added directly to the amplification mixture to tissue blocks. The morphological changes induced by avoid evaporation. With the Gene-Amp In situ PCR System freezing, and the increase rate of diffusion of the amplified 1000 (Perkin-Elmer Corp., Norwalk, CT, USA, product, make frozen sections less desirable for In situ PCR

    purposes. Although thicker sections may compromise http://www.perkin-elmer.com/), up to 3 samples/slide can

    evaluation of morphology and increase noise to signal ratio, it be tested by clamping slides individually using water-tight

    is believed that thick paraffin sections contain more starting seals. Several cheaper ovens, in which In situ PCR can be nucleic acid material and allow a better yield of amplified performed, are also available. The BioOven from Biotherm signal. Deparaffinization is performed on sections cut from Corp. (Fairfax, VA, USA), ensures temperature control via a paraffin blocks, according to standard procedures by sensor placed under the coverslip or glass slide that sends removing paraffin in xylene and rehydration in decreasing information to the control unit. Those that already have a concentrations of ethanol. conventional solution PCR thermocycler can also perform In situ PCR experiments by placing slides in a "tube" block. In 3.4 Fixation (appendix 2) these systems, however, heat transfer to the tissues is less The goal of fixation is to maintain optimal tissue reliable. morphology while preserving the integrity of the nucleic acids. It is generally agreed that aldehyde-based fixatives, such as 3.2 Technical aspects 10% neutral buffered formalin, are superior for preservation The same precautions that apply to solution PCR of morphology and for minimizing the diffusion of signal (17-procedures, should be exercised in In situ PCR experiments. 20). This is fortunate since the vast majority of archival Due to the extreme amplification capability of the In situ PCR, material in pathology departments has been fixed in 10% any small amount of contaminant DNA in the sample can be neutral buffered-formalin. Nevertheless, not all routine inadvertently amplified. During specimen handling, areas procedures for fixation of specimens are done under ideal where amplified DNA (in particular previously amplified standard conditions and, for most protocols, a variety of DNA) can be found should be avoided. Devices (positive proteolytic digestion conditions has to be tested. This is due, displacement pipets, pipet tips containing sterile filters, tubes, in part, to cross-linkages between proteins, or between etc.) have to be specifically allocated to In situ PCR histones and DNA, which are difficult to remove and may experiments and frequent changes of gloves have to become impair the efficiency of PCR. Furthermore, aldehyde-based routine. When working with tissue sections, appropriately fixatives may induce single-stranded breaks that, when coated slides (i.e., poly-L-lysine, Aminosilane) should be used repaired by the DNA polymerase, result in false positivity. to avoid the detachment of sections during the proteolytic Fixatives containing picric acid (Bouin's solution) or mercury digestion step (16-17). To avoid evaporation, a variety of (Zenker's solution) seem to degrade nucleic acids and methods have been attempted. The section is overlaid with generally are not recommended (17-22). Tissues, preferably the PCR mixture and a coverslip is placed over the section. less than 0.5 cm in thickness, are fixed at room temperature The coverslip may be sealed with nail polish or rubber cement in 10 % Neutral Buffered Formalin. Most cell suspensions making sure not to introduce bubbles. After amplification, and cytology preparations are routinely fixed in 95 % alcohol the seal is peeled-off with forceps and the coverslip removed o for 1 hour either at 4C or at room temperature. They can be in 0.1X SSC. Alternatively, specially designed cones (1, 8) o washed in PBS and kept at 4C for short-term storage or are applied to the slide sealing the area of interest. The o dehydrated and placed at 70C for long-term storage. amplification solution is added to the cones making sure that Paraformaldehyde also provides excellent results, being the it diffuses evenly over the tissue section without creating air fixative of choice in many laboratories (21, 22). It can be bubbles. If the operator does not have previous experience used at concentrations ranging from 1 % to 4 % during a with the procedure, it is advisable to attempt several dry runs o period of 4 to 24 hours, preferably at 4C. Fixation has to to get acquainted with the peculiarities of the technique (slide be initiated as early as possible to reduce the chance of manipulation, microscope checks, rubber cement and nail

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In Situ PCR; An Overview

    nucleic acid degradation. or primers, the concentration of the enzyme may vary. It is

     recommended to test concentrations ranging from 0.5 to 5

    units/100 microliter. 3.5 Proteolytic Digestion (appendix 3)

     Since fixatives cross-link proteins and induce other

    changes that diminish the accessibility of reagents to the 4.2 Deoxynucleotide triphosphates (dNTPs) (appendix 5)

    target, pre-treatment with proteases is necessary, particularly Equivalent concentrations of all four dNTPs in the for paraffin-embedded, formalin-fixed tissues (17-19, 21-23). range of 20-200 microM are recommended (25-30). Stringent control over protease digestion is required to Mispriming may be minimized, and sensitivity maximized, by maintain preservation of morphology and to reduce noise to reducing the concentration of the dNTPs (26-30). Typically, signal ratio. Therefore, a balance has to be achieved to avoid in a 100 microliter reaction, a dNTP concentration of 20 false negativity, due to poor penetration of reagents in microM, will allow the synthesis of 2.6 micrograms of DNA underdigested samples, and destruction of the tissue or 10 pmol of a 400 bp sequence (25-27). architecture due to over-digestion by protease. It is

    considered that the length of proteolytic digestion correlates 4.3 Buffer conditions (appendix 5)

    with the duration of fixation (8, 19). As a guideline, 10 min It is important to include the appropriate metallic of digestion is necessary if tissue was fixed for 4 hours in ion in the amplification buffer and to use the enzyme at 10 % neutral buffered formalin. On the other hand, 90 min optimal pH. In general, as compared with solution PCR, 2+of protease digestion may be necessary if tissue was fixed for ions increased concentrations of DNA polymerase and Mg

    are required. This is probably related to the sequestration of 24 h. Bagasra et al. (15), have reported that the presence of

    reagents on slides or due to the presence of inhibitors in the “peppery dots”, observed on the membrane of digested cells,

    tissue sample (24). The concentration of ions is one of the can be used as evidence of appropriate digestion for In situ

    critical factors in PCR experiments and it has to be optimized PCR. In all cases, immediately after digestion, the enzyme to the particular assay conditions. MgClis commonly used should be inactivated. 2at a concentration of approximately 2-5mM.

     3.6 Additional pre-treatments (appendix 4)

    4.4 Primers After proteolytic digestion, additional pretreatment

    Primers are usually designed to be 18-28 steps may be necessary. When labeled nucleotides are used,

    nucleotides in length, with a balanced G/C and A/T ratio, no reduction of the static charge of tissue sections can minimize

    complementarity between their 3' ends (to avoid primer-nonspecific binding. This can be accomplished by washing

    dimer formation) and with a low probability of internal the slide for five minutes in a solution of 0.1M

    secondary structure (23-26). Concentrations between 0.1 and Triethanolamine and 0.25 % acetic anhydride. If peroxidase

    1.5 microM are generally optimal. The melting temperature is going to be used in the detection step, endogenous

    (T) of oligonucleotides ranging in length from 14-70 bases peroxidase should be quenched by any of the routinely used mo can be calculated acording to the following formula: T in C methods. In In situ RT-PCR it may be necessary to pre-digest moo= 2C (#A+#T) + 4C (#G+#C) (24). the sample with RNase-free DNase, to destroy endogenous DNA.

    4.5 Cycling Profile (appendix 5)

     For optimal binding of the primers to the DNA, 4. POLYMERASE CHAIN REACTION (PCR) separation (denaturation) of the two strands has to be complete. Incomplete "denaturation" is a common cause of Prior to attempting In situ PCR protocols, it is PCR failure (25-30). Typically, denaturation is achieved at important to have an appropriate level of knowledge and o o 95C for 30 seconds or at 97C for 15 seconds, although expertise in solution PCR methodologies. This is due to the higher temperatures may be necessary for G+C rich targets. fact that the conceptual background is the same for both Higher temperatures and longer times may lead to the loss of techniques. It is also highly desirable to test reagents and

    enzyme activity since the half-life of Taq polymerase is conditions by PCR in solution (in vitro) as part of the o o approximately 5 minutes at 97C, and 40 minutes at 95C necessary preliminary control process. It is of no use to (24, 28-30). Annealing temperature and annealing time spend time and resources optimizing In situ PCR if one has depend upon the base composition and length and not confirmed that primers and amplification conditions work concentration of the primers (26-30). It is generally agreed as expected, preferably on the nucleic acids extracted from o that an annealing temperature of 5C below the true T of mthe sample. the amplification primers is appropriate and temperatures in o o the range of 55C to 72C yield the best results. The primer 4.1 Polymerases (appendix 5) o extension step (elongation) is usually performed at 72C for A variety of polymerases with different properties 20 seconds to one minute. Using typical concentrations of are commercially available. Ideally, the selected enzyme primers (0.2 microM), annealing will require a few seconds. should work effectively at a given temperature and retain its o However, a longer extension time may be required at the C . It is enzymatic activity at temperatures higher than 95beginning of the cycling when the substrate concentration is important to follow the conditions suggested by the supplier, low or at later cycles when substrate concentration exceeds since, depending on the vendor, the same enzyme may have the enzyme concentration. Some investigators report that different assay requirements. In general, 0.5-2.5 units of longer extension times (e.g., 2 min) are necessary (30) while DNA Taq polymerase in a 100 microliter reaction is others omit the extension step since annealing seems to be the appropriate (21-24). However, based on the type of targets

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In Situ PCR; An Overview

    rate limiting factor (2, 31-33). The reaction is maintained at 5.2 Direct In situ PCR (appendix 7) o C for 5-15 minutes to allow completion of partial 72In direct In situ PCR, the label, digoxigenin or extension and annealing (25-29). It is believed, however, that biotin, is incorporated, into the amplified product, during the In situ DNA amplification occurs at a low efficiency. It is PCR step (32-36). Digoxigenin is present in Digitalis plants generally estimated that in 30 cycles, even under the best and therefore is not detectable in any other biological material. conditions, amplification of DNA does not exceed 50-100 High affinity, unconjugated, anti-digoxigenin antibodies and fold, in suspended cells, and it may be even lower on tissue anti-digoxigenin antibodies conjugated to alkaline sections and cytospins (20-23). The reasons are not clearly phosphatase, peroxidase, fluorescein or rhodamine, are understood although cross-linking of histones to DNA, single-readily available (37). Biotin is a member of the vitamin B stranded DNA breaks, and sequestration of DNA complex and can be detected with an anti-biotin antibodies polymerases and other reagents on the surface of slide, have (37). The final result is the enzymatic localization of a all been proposed as potential causes (24). When all chromogen at the site of DNA amplification. Due to its parameters are optimized, the number of cycles will primarily simplicity, direct In situ PCR represents a logical procedure of depend upon the starting concentration of DNA (26-30). It choice. However, in the experience of many investigators (5, has been estimated that if the starting number of target 8, 24, 35), direct detection procedures tend to yield false-molecules is 300,000, then 25-30 cycles of amplification positive results (35-36) that may be due to either nonspecific would be appropriate. On the other hand, if only 50 target incorporation of labeled nucleotides into fragmented molecules are present, 40-45 amplification cycles may be endogenous DNA undergoing “repair” by the DNA necessary (30-32). Some investigators, however, reported polymerase (DNA-repair artifact), or nonspecific priming by poor results, with more than 20 cycles, in In situ PCR cDNA or DNA fragments (endogenous priming) (35-36). experiments (4). The false positive signal is typically present in the nucleus,

     particularly in apoptotic and senescent cells where DNA 5. IN SITU PCR PROTOCOLS fragmentation occurs (26, 29-31). This endogenous DNA

     amplification is difficult to avoid, even by DNAse

    In “direct” methods, labeled nucleotides (i.e., pretreatment (5, 35, 36). Several alternatives to reduce the digoxigenin-11-dUTP, fluorescein-dUTP) are incorporated artifact have been attempted with only partial success. These during the amplification step and then amplified product are include the use of exonuclease-free DNA polymerase (5, 36-detected by immunohistochemistry. In “indirect” methods, 38), repair of DNA nicks by treatment with T4 ligase (39-44), the amplified products are detected by In situ Hybridization or initial thermal cycling using unlabeled nucleotides (44, 48). (ISH) using a specific labeled probe.

     5.3 Indirect in situ PCR (appendix 8-9)

    5.1 In situ RT-PCR (appendix 6) The indirect method, although more cumbersome,

     Since mRNA cannot serve as a template in PCR, provides an extra level of specificity since the probe is a reverse transcription step is introduced to convert designed to be complementary to an internal sequence within mRNA to cDNA (1-10). The combination of these two the amplified product (39, 48, 49). Double-stranded DNA, techniques is referred to as RT-PCR. RT-PCR may be single-stranded DNA, oligonucleotides 20-30 bases long, and used for the detection of mRNAs that are present at less single-stranded RNA have all been successfully used as than 10 copies/cell (1-12). The enzyme that converts probes for the detection of amplified products (30). RNA into c-DNA is "Reverse Transcriptase". Three Hybridization of probes to the amplified product follows the Reverse Transcriptases commercially available are: the general rules described for In situ Hybridization (ISH) mesophilic viral reverse transcriptases from Avian procedures (50). The kinetics of the reaction are Myeloblastosis Virus (AMV) and Moloney Murine influenced by: a.) accessibility of the target (in In situ Leukemia Virus (M-muLV), and rTth, a heat-stable DNA PCR methods, the target is readily accessible because polymerase from Thermus thermophilus, that can be used hybridization takes place after DNA amplification), b.) to perform reverse transcription and PCR in a single step. concentration of the probe, and c.) stringency of The AMV and M-muLV RTs can reverse transcribe hybridization. A series of parameters influence the mRNAs up to 10 kb; rTth synthesizes cDNAs in the stability of the hybrids: a.) T (formamide decreases range of 1-2 kb. The most important factors to consider min the selection of the enzyme are: a.) RNAse H activity the T allowing use of lower temperatures to achieve a mthat degrades RNA in an RNA:cDNA hybrid, b.) higher stringency), b.) base composition (the greater o temperature (M-muLV reaches maximum activity at 37C the G+C content the higher the T), c.) sequence mo and AMV at 42C); rTth reverse transcribes mRNA at identity between the probe and the target, and d.) o o 60-70C and amplifies cDNA at 60-94C), and c.) composition of hybridization/washing solution (higher divalent ion requirements (rTth requires manganese). concentrations of monovalent cations increase the Three types of primers can be used in reverse stability of hybrids). The final sensitivity depends on transcription reactions: a.) Oligo(dT)12-18 (it binds to the the method of probe labelling and detection. Labelling endogenous poly (A)+ tail of mRNA), b.) random 3of the probe can be done with radioactive labels ([H], hexanucleotides (these bind at any complementary 353233sequence throughout the length of mRNA), c.) specific [S], [P], [P]. Factors to be considered are cost, oligonucleotides (based on a specific mRNA sequence). instability, and biohazard potential of the radioisotope

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    In Situ PCR; An Overview

    DNA polymerase (44). Excessive protease digestion used. Alternatively, the probe can be labeled with non-(removal of histones), insufficient DNAse treatment (DNase radioactive haptens, such as biotin, digoxigenin or may break DNA into oligonucleotides), poor fixation, FITC. The efficiency of the non-radioactive detection suboptimal processing of the sample, apoptosis, or previous methods has been recently improved using irradiation of the specimen can all induce DNA breaks. The immunogold silver detection methods (38, 51). slide, in which the DNA polymerase is omitted, however, should not show this artifact. This type of false positivity is 6. CONTROLS extremely difficult to eradicate but it may be partially reduced by using, exonuclease-free, DNA polymerase or by In In situ PCR protocols, it is imperative to performing several amplification cycles in the absence of the control the numerous steps involved, not only to labeled nucleotide (44, 45). interpret a given signal in the proper context of sensitivity and specificity, but also to identify and 6.4 Mispriming. correct some of the many potential pitfalls that are The frequent amplification of "non-desired"

    likely to occur (appendix 6). sequences seems to be dependent upon factors such as

    specificity of primers, pH and ion concentration in the PCR mixture, and annealing temperature (1-8). Primers should be 6.1 Causes of false negativity. designed to detect short template sequences avoiding Poor thermal conduction by the slide or the block, homology to non-desired sequences and between themselves. uneven convection, adsorption of reagents to the glass, Since misprimimg occurs at lower melting temperatures, presence of DNA polymerase inhibitors, evaporation, owithholding the Taq polymerase, until 55 C is reached excessive washing, and leakage of reagents, can all account reduces non-specific amplification without compromising the for false negativity or inconsistency in the results (43-48). specific primer-target annealing. This "hot-start" procedure Control for some of these steps should be done during the has been successfully applied to In situ PCR (46-48). An optimization of the technique. Since most In situ PCR alternative method has been developed which takes advantage protocols are designed to detect nucleic acids within cells of the affinity interaction of Taq and Anti-Taq mAb (46). placed on slides, the starting material may be present in low This interaction initially blocks the activity of the enzyme but amounts (43-45) and uncontrolled fixation of archival the complex dissociates, at higher temperatures, freeing the material may have further reduced the nucleic acid content, enzyme at the appropriate time for specific amplification. In compromising the sensitivity of the procedure. DNA the hands of some researchers, this method has provided extracted from paraffin blocks is often shorter than 400 bp results similar to hot-start technique with less manipulation of and mRNA fragments, in paraffin blocks, are usually smaller

    the sample (40-42). Other “cold-start” procedures, using E. than 200 bp (1-10, 43-45). Although, some authors have

    coli single-strand-DNA-binding protein SSB, or T4 gene been able to obtain full length DNA from paraffin-embedded

    protein, at ambient temperatures, have been devised (1, 5, 8, material (2, 12-14), other investigators have succeed with In

    40, 42, 48, 50-53). situ PCR only when using multiple primers designed to

     produce short PCR products and relatively long DNA probes

    6.5 Endogenous priming. or cocktails of oligonucleotide probes (8, 43-45). To ensure This artifact, thought to occur due to priming of that a negative result is not due to poor penetration of endogenous DNA and cDNA fragments, is difficult to reagents and accessibility of the target, it is advisable to eradicate and therefore the use of specific primers is include a control sample in which a sequence, such as actin, imperative (1, 8). is amplified.

    6.6 Diffusion of the PCR products. 6.2 Causes of False Positivity. Excessive protease digestion (loss of cellular Specificity can be tested by recovering the product boundaries) and excessive number of amplification cycles from the slide and analyzing it by gel electrophoresis. In may contribute to the diffusion of the amplified product (8). indirect methods, the ISH step introduces an extra level of If a product diffuses out of its original location, it may be specificity, determined by the probe. Furthermore, another preferentially amplified, producing a signal in a different slide hybridized with an irrelevant probe should give no signal. cellular compartment or even in an extracellular location with "Nested" PCR can increase the specificity of PCR by using a the possibility of "labeling" adjacent negative cells (8, 12, 21). second set of primers to amplify sequences within the first Proper processing of the sample, use of aldehyde-based amplicon (46-48). Furthermore, testing, in the absence of fixatives, low number of cycles, generation of longer PCR components (enzyme, primers or probe), should be amplicons or more “bulky” products (21, 41), or the use of done to make sure that the detection system does not render a direct methods (incorporation of digoxigenin) may all reduce non-specific signal. In experiments with virally-infected cells, this type of false positivity (1, 4, 12, 48). it is advisable to mix infected and non-infected cells, at different ratios, and to correlate the results of the test with the

    7. APPLICATIONS expected sensitivity.

     Despite technical difficulties, In situ PCR protocols 6.3 DNA-repair. are being utilized in an increasing number of applications. Non-specific incorporation of labeled nucleotides

    The technique has particular potential in areas such as may occur as a consequence of "repair" of DNA breaks by

20

In Situ PCR; An Overview

    embryogenesis, organogenesis, infectious diseases, genetics, Obstacles to the wide application of In situ PCR

    immunology, neoplasia or pathology. New genes are have been low amplification efficiency, poor reproducibility, continuously being added to the list of target sequences, and difficulty in quantitation (21). Improvements in the detected by In situ PCR, that includes infectious agents such technology, however, are likely to reduce these drawbacks. as human immunodeficiency virus (HIV-1), simian Newer reported systems, such as the nanogold-silver immunodeficiency virus (SIV), human papilloma virus detection (51) or the catalyzed reporter deposition methods (HPV), hepatitis B virus (HBV), cytomegalovirus (CMV), (37) may reduce background. Another innovative procedure Epstein-Barr virus (EBV), human herpes virus-6 (HHV-6) takes advantage of a fluorochrome-labeled oligonucleotide and herpes simplex virus (HSV), and molecules such as p53, probe, specific for a region of the amplified DNA between the surfactant protein A, estrogen receptor, growth factors, PCR primer sequences, that is designed to emit fluorescence growth factor receptors, transferrin, and adrenomedullin (88-signal only after hybridizing to the appropriate template (4,21). 110). Almost every tissue has been successfully tested (3, 48-

    57). It is expected that In situ PCR protocols will be

     further simplified in the near future and that this methodology

    One of the areas where In situ PCR methodology and its variants, despite limitations, will find a deserved place has proven its value is the detection of viral sequences that in the repertoire of methods available to the researchers. are present in vanishingly small numbers (48-57). This is

    particularly true for slowly progressing viral diseases. Among 8. ACKNOWLEDGEMENT

    these are the group of lentiviruses of which HIV is a member

    This work was supported in part by the American Cancer where In situ PCR has provided a ten-fold increase in

    Society Institutional Research Grant # 202 sensitivity over the conventional techniques (51-53).

     Furthermore, the Creutzfeld-Jacob virus of Progressive

    Multifocal Leukoencephalopathy has been demonstrated by 9. REFERENCES

     In situ PCR in an archival material from 1958, confirming

    1. Teo IA, Shaunak S. Polymerase chain reaction In situ: previous solution PCR data and demonstrating that DNA

    sequences are preserved for long periods of time in routinely an appraisal of an emerging technique. Histochem J 27,

    647-659 (1995) processed tissues (57). The increased sensitivity of In situ

    PCR offers the opportunity for viral latency studies, 2. Nuovo GJ. PCR In situ Hybridization. Protocols and

    applications. Ed: Nuovo GJ, Raven Press. New York, 1992 overcoming the need to activate the viruses in vitro prior to

     detection. For example, Herpes Simplex Virus was detected

    3. Embleton M , Gorochov J, Jones PT, & Winter G. In-in the trigeminal ganglia of latently infected mice. In situ

    cell PCR from mRNA: Amplifying and linking the rearranged PCR revealed three times more infected neurons than it was

    immunoglobulin heavy and light chain V-genes within single shown previously (56).

    cells. Nucleic Acids Res 20, 3831-3837 (1992)

     A fast, alternative method to In situ Hybridization,

    4. Patterson B, Till M, Otto P, Goolsby C, Furtado M, Primed In situ DNA Synthesis (PRINS) (60, 61) involves McBride L, & Wolinsky S. Detection of HIV-1 DNA and annealing of oligonucleotides or short DNA fragments to

    messenger RNA in individual cells by PCR-driven In situ denatured complementary nucleic acid sequences (i.e.,

    hybridization and flow cytometry. Science 260, 976-979 metapahase or interphase chromosomes) on slides. A

    (1993) thermostable DNA polymerase incorporates nonradioactive

     labeled nucleotides that are detected by immunofluorescence.

    5. Komminoth P, Adams V, Long AA, Roth J, Saremaslani A variety of suitable primers for chromosomal sequences are

    P, Flury R, Schmid M, & Heitz PU. Evaluation of methods available. A further increase in sensitivity has been achieved

    for hepatitis C virus (HCV) detection in liver biopsies: with Cycling PRINS. To detect low copy or unique

    sequences, the PRINS technique can be modified by comparison of histology, immunohistochemistry, In situ

    subjecting the chromosomal preparation to amplification hybridization, reverse transcriptase (RT) PCR, and In situ RT

    cycles that incorporate biotin or digoxigenin-labeled PCR. Path Res Pract 190, 1017-1025 (1994)

    nucleotides. Detection can be accomplished by

    immunofluorescence with suitable antibodies. 6. Chen RH, Fuggle SV. In situ cDNA polymerase chain reaction. A novel technique for detecting mRNA expression. A related technique, Self-Sustained Sequence Am J Pathol 143, 1527-1534 (1993) Replication-Based Amplification (3SR) (63, 64) is a reiterated cycling of reverse transcription and amplification 7. Komminoth P, Long AA. In situ polymerase chain reactions, catalyzed by AMV-RT and T7 RNA polymerase, reaction. An overview of methods, applications and intended to replicate a RNA target via RNA/DNA hybrids and limitations of a new molecular technique. Virchows Arch B double-stranded cDNA intermediates. The cDNA copy of the 64, 67-73 (1993) original mRNA incorporates a T7 RNA polymerase promoter that is used as template for a 10 million fold amplification 8. Long AA, Komminoth P, Lee E, & Wolfe H. within 2 h. The destruction of RNA in RNA/DNA hybrids Comparison of indirect and direct In situ polymerase chain by E. coli RNAseH eliminates the need for denaturation steps. reaction in cell preparations and tissue sections. Detection of viral DNA, gene rearrangements and chromosomal

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In Situ PCR; An Overview

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