Molecular Diagnosis of Genetic Diseases Second Edition Edited by Rob Elles, PhD Roger Mountford, BSC M E T H O D S I N M O L E C U L A R M E D I C I N E TM Molecular Diagnosis of Genetic Diseases Second Edition Edited by Rob Elles, PhD Roger Mountford, BSC 1 1 Optimizing PCR for Clinical Diagnosis Michael P. Bulman 1. Introduction The polymerase chain reaction (PCR) has rapidly become an essential tool within the diagnostic laboratory. Therefore, it is crucial when setting up a new PCR-based test to ensure that the PCR reaction is carefully designed to be as robust and reliable as possible. Usually, little optimization is required. However, there are some instances when a particular region of DNA proves difficult to amplify by PCR. A number of factors are important to consider when choos- ing PCR conditions, and these are discussed in this chapter. 2. Materials Analytical-grade reagents should be used at all stages, unless otherwise stated. 2.1. PCR Reaction Buffers As an alternative to the buffer supplied by the manufacturer, use of either of the following buffers may be beneficial in the PCR. 2.1.1. Buffer “A” 10X 1. 670 mM Tris-HCl (pH 8.0 at 25°C). 2. 166 mM ammonium sulfate. 3. 37 mM magnesium chloride. 4. 67 µM ethylenediaminetetraacetic acid (EDTA). 5. 0.85 mg/mL bovine serum albumin (BSA). Filter-sterilize and store in 1-mL aliquots at –20°C. This is a particularly robust PCR buffer, which provides a good yield of prod- uct, and is excellent regardless of the quality of the DNA template. From: Methods in Molecular Medicine, vol. 92: Molecular Diagnosis of Genetic Diseases, Second Edition Edited by: R. Elles and R. Mountford © Humana Press Inc., Totowa, NJ CH01,1-8,8pgs 8/11/03 12:28 PM Page 1 2.1.2. Buffer “R” 20X 1. 1.0 M Tris-HCl (pH 9.0 at 25°C). 2. 400 mM ammonium sulfate. 3. 30 mM magnesium chloride. Filter-sterilize and store in 1-mL aliquots at –20°C. This buffer is useful for templates that are difficult to amplify, such as those with GC-rich tracts, and for longer templates (800 basepairs [bp] + in size). 2.2. Deoxynucleotide Triphosphates (dNTPs) Purchase individual dNTPs as 100 mM stocks (e.g., Roche Diagnostics UK Ltd, Lewes, UK; dNTP set #1969064). Make a 2.5-mM working stock by com- bining 2.5 µL of each dNTP and adding 90µL of water. Store dNTPs in small aliquots at –20°C and avoid multiple freeze-thaw cycles, as dNTPs are prone to degradation (especially for multiplex PCR). The final concentration of each dNTP in the PCR reaction is usually 200–250 µM. To increase the speci- ficity/fidelity of the PCR, decrease the concentration of dNTPs with a corre- sponding decrease in MgCl 2 concentration. 2.3. Magnesium Chloride (if required) 10X “A” and 20X “R” buffers already contain magnesium at 37 mM and 30 mM respectively (to give a final concentration in the PCR reaction of 3.7 mM and 1.5 mM,respectively). However, for those 10X buffers that do not contain magnesium, this is usually supplied as a 25–50-mM stock and should be used at 1.5–5.0 mM final concentration in the PCR. Mix the magnesium thor- oughly prior to addition to the PCR mix. 2.4. Oligonucleotide Primers The final concentration in a PCR reaction should be between 0.1 µM and 0.5 µM of each primer. Primer concentrations that are too high may lead to mis- priming in the reaction. Conversely, primer concentrations that are too low may not give good yields of product. 2.5. Polymerases There are many suppliers and varieties of heat-stable Taq polymerases on the market. (See http://www.alkami.com/methods/refpoly.htm and/or http:// www.neb.com/neb/frame_tech.html for information of most of those avail- able.) The most frequently used enzyme is Taq polymerase. This enzyme does not have a 3′–5′ exonuclease activity, which has two consequences: it exhibits a non-template addition of usually an adenine base at the 3′ end of the product (1); secondly, the lack of the 3′–5′ exonuclease activity means that Taq does not correct for the incorporation of mismatched bases—it has no “proof- 2 Bulman CH01,1-8,8pgs 8/11/03 12:28 PM Page 2 reading” activity (2–5). Therefore, for those techniques such as PCR-based site- directed mutagenesis where a reduced error rate is crucial, it is important to use a proof-reading enzyme such as Pfu from Stratagene (La Jolla, CA) (6) or Vent ™ polymerase from New England Biolabs (Hitchen, Hertsfordshire, UK) (7). For the vast majority of applications, however, Taq is perfectly adequate. 2.6. Template DNA Dilute good-quality genomic DNA in TE or deionized water to approx 25–50 ng/µL and use 2.5-µL in a 25-µL reaction. 3. Methods Unfortunately, there is no single set of conditions that can be applied to all PCR amplifications. Factors such as primer sequence, product length, and primer annealing temperature will differ for each assay. For the reliable ampli- fication of a specific target, the optimal conditions for PCR will be found empirically. However, a well-designed PCR reaction should work with little or no optimization necessary. 3.1. Design of PCR Primer Pairs The selection of the correct pair of primer sequences for the PCR reaction may be the most critical parameter for successful PCR. The primer set must hybridize efficiently to the target sequence with as little hybridization as pos- sible to other sequences also present in the sample. Poorly designed primers may result in the synthesis of little or no product as a result of “primer-dimer” formation and/or nonspecific amplification. 3.2. Primer Length In general, oligonucleotides between 20 and 30 bases are sufficiently sequence-specific for complex genomes, provided that the annealing tempera- ture is optimal. 3.3. GC Content Ideally, primer sequences should be designed to have a GC content between 45% and 55%. Stretches of poly C or poly G should be avoided, as these can promote nonspecific annealing. Similarly, stretches of poly A or poly T should also be avoided, as these may open up stretches of the primer-template complex. 3.4. Melting Temperature ( T m ) As a starting point, the annealing temperature for a primer pair is calculated as 5°C below the estimated T m . Ideally the primers should closely match each other in their melting temperatures, or amplification efficiency will be reduced and may even lead to the failure of the PCR. Optimizing PCR for Clinical Diagnosis 3 CH01,1-8,8pgs 8/11/03 12:28 PM Page 3 A rough and ready way to calculate T m for primers <20 bp is (8): T m = [4(G+C) + 2(A+T)]°C (1) To calculate T m for primers >20 bp use (9): T m = 62.3°C + 0.41°C (%G-C) – 500/length (2) Online T m calculators such as that found at http://alces.med.umn.edu/ rawtm.html are also extremely useful. 3.5. General Comments on Primer Design Alternatively, use a free web tool such as Primer3 (available at http://www- genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) or a commercially avail- able program such as Oligo 6 primer analysis software (Molecular Biology Insights Inc., Cascade, CO; http://www.oligo.net) to help design primers. In conclusion, an “ideal” primer will have a 50% GC content with a near random nucleotide composition, and will be 20–25 bp long resulting in a T m of 56–62°C. However, primers can only be designed from the available sequence, and sometimes primer design is tricky. Compromise is the key, and it is very unusual to be unable to design a primer pair that will work after some optimization. 3.6. Standard PCR Reaction A standard 25µl PCR reaction contains the following components: Component Volume 10X buffer 2.5 µL dNTP mix 2.5 mM each 2.5 µL 5 µM primer mix 5.0 µL Taq polymerase (5 U/µL) 0.05–0.2 µL DNA template (~25–50 ng/µL) 2.5 µL Sterile deionized water to 25 µL It is important to mix all components thoroughly after thawing prior to assembly of the PCR master mix. Note: It is essential to include a water “negative control” for each PCR setup, otherwise contamination in the reaction components is not apparent. 3.7. PCR Program 3.7.1. Denaturing Temperature This is normally 94–95°C for 30 s to 1 min per cycle after an initial 3–5 min incubation at 95°C. It is important to keep the denaturation temperature/ time to a minimum, since Taq polymerase may lose activity in repeated cycling. 4 Bulman CH01,1-8,8pgs 8/11/03 12:28 PM Page 4 3.7.2. Annealing Temperature See Subheading 3.4. to estimate the T m and anneal at 5°C below this temperature. 3.7.3. Elongation Temperature and Time The elongation temperature is normally 70–72°C, and the elongation time depends on the size of the final size of the PCR product. Generally, 30 s to 2 min is sufficient for most PCR reactions; however, for larger products, a general rule is to extend for 1 min per kilobase of product size. 3.7.4. Cycle Number The standard number of cycles necessary for efficient amplification is 25–40 cycles. Increasing this to >40 cycles does not generally increase the rel- ative amount of PCR product because of the plateau effect, in which the expo- nential rate of product accumulation in the later stages of PCR is attenuated (10). 3.7.5. Final Extension Program one final extension cycle for 7–10 min at 72°C to fill in any incom- plete polymerization. Then cool the reaction to 4–18°C. 3.7.6. Thermal Cyclers Many thermal cyclers are available. Some of these are compared at http:// www.biocompare.com/molbio.asp?catid=33. The author uses the 0.5-mL GeneAmp ® PCR System 9700 (Applied Biosystems [ABI], Foster City, CA) and the MJ Research DNA Engine Tetrad ™ Thermal Cycler (Genetic Research Instrumentation Ltd, Braintree, Essex, UK). 4. Notes Most manufacturers of PCR reagents and equipment have excellent web sites with useful online guides that can often be downloaded as PDFs (e.g., Qiagen at http://www.qiagen.com/literature/pcrlit.asp; ABI at http://www. appliedbiosystems.com/support/techtools/; Invitrogen at http://www.invitrogen. com/content.cfm?pageid = 4155&cfid = 6398854&cftoken = 97182355). 4.1. Hot Start PCR PCR hot start can be used to increase the reaction sensitivity, reduce non- specific products in the PCR, and increase the PCR yield. The simplest way to set up hot start PCR is to use one of the chemically modified Taq polymerases Optimizing PCR for Clinical Diagnosis 5 CH01,1-8,8pgs 8/11/03 12:28 PM Page 5 such as AmpliTaq Gold (ABI). For details, see http://www.appliedbiosystems. com/products/productdetail.cfm?ID = 104 or Platinum Taq (Invitrogen Ltd., Paisley, UK). 4.2. Enhancers for PCR It may be beneficial to use one or more additives to increase the yield, specificity, and consistency of the PCR reaction. A variety of such agents are available, including dimethyl sulfoxide (DMSO), dimethyl formamide, betaine (N, N, N-trimethylglycine = [carboxymethyl] trimethylammonium) formamide, 7-deaza-2′-deoxyguanosine (7 deaza GTP), non-ionic detergents (e.g., Triton X-100, Tween 20, and Nonidet P-40), BSA, urea, and glycerol. These additives are believed to lower the T m of the target DNA. A helpful discussion of the benefits of the most useful additives can be found at Rob Cruickshank’s PCR additive page at http://taxonomy.zoology.gla.ac.uk/ ~rcruicks/additives.html Two of the most commonly used PCR additives are the organic solvents DMSO and betaine. Both of these are particularly useful for GC-rich templates by acting as helix destabilizers. DMSO is included in the PCR reaction at a final concentration of 5–10% (v/v), although DMSO at 10% and higher has been shown to reduce the activity of Taq by up to 50% (10,11). Betaine is used at a final concentration of 1 M (from a 5-M stock in water), and is often included in commercial PCR kits as an “unidentified” additive. Recently, a number of novel potent PCR enhancers have been discovered, and the most effective of these is tetramethylene sulfoxide (12). Use of low- mol-wt compounds such as this has been shown to be more beneficial in the amplification of high GC-rich templates than DMSO and betaine. 4.3. PCR Mixture for Difficult to Amplify Templates Compound Volume 10X PCR buffer 2.5 µL Modified dNTP mix * 1.0 µL 5 µM primer mix 5.0 µL 1 mM 7-deaza GTP 1.25 µL DMSO 1.25 µL 5 M betaine 5.0 µL Amplitaq Gold ™ 0.25 µL DNA (25 ng/µL) 5.0 µL Sterile deionized water To 25 µL * Modified dNTP mixture contains: 5 µL each of dTTP, dCTP, and dATP plus 3.8 µL of dGTP made up to 100 µL with water (100-mM stocks of each dNTP). 6 Bulman CH01,1-8,8pgs 8/11/03 12:28 PM Page 6 Cycling conditions: 1. 95°C * 12 min 1 cycle 2. 95°C * 11 min 3. X°C * 11 min 40 cycles 4. 72°C * 12 min 5. 72°C * 10 min 1 cycle * Annealing temperature varies depending on the primer set. 4.4. Multiplex PCR This is a demanding technique often used to amplify several PCR products in a single reaction. Extensive optimization is often required to produce a robust and reliable PCR reaction as with multiple primer pairs in a single-tube reac- tion, which increases the likelihood of primer-dimer and other nonspecific products that may interfere with the amplification of the products required. An extensive troubleshooting guide for multiplex PCR can be found at http://www. info.med.yale.edu/genetics/ward/tavi/Trblesht.html. References 1. Clark, J. M. (1988) Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 25, 9677–9686. 2. Tindall, K. R. and Kunkel, T. A. (1988) Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 9, 6008–6013. 3. Krawczak, M., Reiss, J., Schmidtke, J., and Rosler, U. (1989) Polymerase chain reaction: replication errors and reliability of gene diagnosis. Nucleic Acids Res. 25, 2197–2201. 4. Kwok, S., Kellogg, D. E., McKinney, N., Spasic, D., Goda, L., Levenson, C., et al. (1990) Effects of primer-template mismatches on the polymerase chain reac- tion: human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 25, 999–1005. 5. Eckert, K. A. and Kunkel, T. A. (1991) DNA polymerase fidelity and the poly- merase chain reaction. PCR Methods Appl. 1, 17–24. Review. 6. Lundberg, K. S., Shoemaker, D. D., Adams, M. W., Short, J. M., Sorge, J. A., and Mathur, E. J. (1991) High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 1, 1–6. 7. Mattila, P., Korpela, J., Tenkanen, T., and Pitkanen, K. (1991) Fidelity of DNA syn- thesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity. Nucleic Acids Res. 25, 4967–4973. 8. Suggs, S. V., Hirose, T., Miyake, E. H., Kawashima, M. J., Johnson, K. I., and Wallace, R. B. (1981) Using Purified Genes, in ICN-UCLA Symp. Dev. Biol. 23, 683. 9. Bolton, E. T. and McCarthy, B. J. (1962) Proc. Natl. Acad. Sci. USA 48, 1390–1397. Optimizing PCR for Clinical Diagnosis 7 } CH01,1-8,8pgs 8/11/03 12:28 PM Page 7 10. Innis, M. A. and Gelfand, D. H. (1990) Optimisation of PCRs, in PCR Protocols (Innis, Gelfand, Sninsky and White, eds.), Academic Press, New York, pp. 3–12. 11. Gelfand, D. H. and White, T. J. (1990) Thermostable DNA polymerases, in PCR Protocols (Innis, Gelfand, Sninsky and White, eds.), Academic Press, New York, pp. 129–141. 12. Chakrabarti, R. and Schutt, C. E. (2002) Novel sulphoxides facilitate GC-rich template amplification. BioTechniques 32, 866–874. 8 Bulman CH01,1-8,8pgs 8/11/03 12:28 PM Page 8 9 2 Current and Emerging Techniques for Diagnostic Mutation Detection An Overview of Methods for Mutation Detection Claire F. Taylor and Graham R. Taylor 1. Mutation Detection: An Introduction This chapter provides a broad overview of the range of mutation detection techniques that are now available. For the purposes of this chapter, a mutation can be defined as a sequence change in a test sample compared with the sequence of a reference standard. This definition implies nothing about the phenotypic consequences (e.g., path- ogenicity) of a mutation. A polymorphism may be defined as a mutation that occurs in a substantial proportion (>1%) of a population and is tacitly assumed to be non-pathogenic, although the true pathogenicity may be unknown. A polymorphism has also been defined as a Mendelian trait that exists in the pop- ulation, with the frequency of the more rare of the two alleles greater than 1–2% (1). If we accept that DNA sequence is a Mendelian trait, then the two defini- tions of polymorphism are the same. The detection of a single base change in the human genome requires a signalϺbackground ratio of 1Ϻ6 × 10 9 —a formidable task. To achieve such selectivity in the field of electronics would require amplification and noise reduction, and it is no surprise that analogous processes are found in molecu- lar genetics—for example, amplification by the polymerase chain reaction (PCR) and noise reduction by the stringent annealing of probes and primers. Mutation detection techniques can be divided into techniques that test for known mutations (genotyping) and those that scan for any mutation in a par- From: Methods in Molecular Medicine, vol. 92: Molecular Diagnosis of Genetic Diseases, Second Edition Edited by: R. Elles and R. Mountford © Humana Press Inc., Totowa, NJ CH02,9-44,36pgs 8/11/03 12:29 PM Page 9 [...]... initial scanning for novel mutations in papers published in the journals Nature Genetics, the American Journal of Human Genetics, the Journal of Medical Genetics, Human Molecular Genetics, and Human Mutation All papers that describe mutation scanning and which specified the technique employed were included, regardless of study size or purpose When more than one method was used for primary screening,... polypeptide-chain-terminating mutations When the nature of mutation is unknown, a detection method that is unbiased toward any type of mutation should be used For conditions/genes in which a single type of mutation predominates, it may be more appropriate to select a method designed to detect only that type of mutation 5.4 Features of the DNA Sequence Analyzed Knowledge of the presence of common polymorphisms in the fragment... the region of interest forms the lowest melting domain, a GC clamp of 20–40 bp is usually added to one end of the fragment to be analyzed (135) The sensitivity of DGGE is in the range of 95–100% (136) for fragments of up to 500 bp In classical DGGE, separation is achieved by electrophoresis through a polyacrylamide gel containing a chemical denaturant gradient Variations on the principle of DGGE include... of up to 3.5 kb has also been described (196) 9.2 Functional Assays A small number of assays that directly test protein function from a cloned DNA sequence have been described (197–199) Successful applications of functional assays have been described (see 185, 200) However, applications for functional assays are limited, not least because of the paucity of information about the molecular function of. .. identification of methylated residues in the starting sample (102) Direct sequencing of PCR products yields a strandspecific average of the methylation pattern in the starting population of molecules For information about the methylation pattern of individual molecules, cloning of the PCR products prior to sequencing is required Bisulfite modification can lead to the methylation-dependent creation of novel... detection of mitochondrial DNA deletions: Characterization of a novel deletion Clin Biochem 31, 627–632 74 Voskova-Goldman, A., Peier, A., Caskey, C T., Richards, C S., and Shaffer, L G (1997) DMD-specific FISH probes are diagnostically useful in the detection of female carriers of DMD gene deletions Neurology 48, 1633–1638 75 Worley, K C., et al (1995) Rapid molecular cytogenetic analysis of x-chromosomal... analysis (CSA) (187), a development of orphan peak analysis (188) is an alternative method of analyzing the products, making a direct comparison of mutant and wild-type sequencing data without the use of base-calling software Although sensitivity is high and mutations are defined as well as identified, the limitations that apply to sequencing also apply to CSA Sequencing of heterozygotes by matrix-assisted... change in size of the protein compared to a wild-type control Sensitivity for truncating mutations is high (reviewed in 195) with most false-negative results because of mutations at the ends of the fragment Fragment size for PTT analysis is typically in the range of 1–1.5 kb: for the majority of genes, PTT analysis requires cDNA or large exons as a starting material The biggest advantage of PTT is that... denaturing gel conditions (129) For fragments in the size range of 200–800 bp, sensitivity of 88% has been detected, and a reduction in the maximum size of the fragment has been associated with an increase in the detection rate to 100% (130) Mutations within 50 bp of the end of a fragment are not detected, presumably because the distortion of the duplex is not great enough to generate a significant mobility... limited use of the Affymetrix system for mutation detection thus far lies in its limited sensitivity Di-deoxy sequencing of the p53 gene in 100 primary human lung cancers by cycle sequencing was compared with sequence analysis by using the p53 GeneChip assay (58) The GeneChip assay detected 46 of 52 missense mutations (88%), but 0 of 5 frameshift mutations The specificity of direct sequencing and of the . provides a good yield of prod- uct, and is excellent regardless of the quality of the DNA template. From: Methods in Molecular Medicine, vol. 92: Molecular Diagnosis of Genetic Diseases, Second Edition Edited. Molecular Diagnosis of Genetic Diseases Second Edition Edited by Rob Elles, PhD Roger Mountford, BSC M E T H O D S I N M O L E C U L A R M E D I C I N E TM Molecular Diagnosis of Genetic. (genotyping) and those that scan for any mutation in a par- From: Methods in Molecular Medicine, vol. 92: Molecular Diagnosis of Genetic Diseases, Second Edition Edited by: R. Elles and R. Mountford © Humana