CHAPTER 1 The Polymerase Chain Reaction Getting Started Charles R. M. Bangham 1. Introduction The polymerase chain reaction (PCR) uses two oligonucleotide primers to direct the synthesis of specific sequences of DNA. One primer anneals to the coding strand of DNA and the other to the anticoding strand; the primer binding sites are typically separated by a few hundred base pairs (loo- 1000 bp). Repeated cycles of polymerization and denaturation lead to the exponential increase of the sequence defined by the primers. The extraordi- nary sensitivity and specihcity of PCR have established it as a standard tech- nique in molecular biology in the short time since it was first described (1). The purpose of this chapter is to suggest starting conditions for a PCR reaction and ways to overcome the main problems in PCR. It is intended as a practical guide, so theoretical aspects will not be discussed in detail. For a fuller account, there are excellent and comprehensive guides edited by Erlich (2) and by Innis et al. (3‘). Protocols for special applications of PCR are de- scribed in later chapters in this volume. 2. Choice of Primers and Target DNA Sequence The ideal oligonucleotide primer has the following features: l Length: 18-30 bp. Shorter and longer primers may, however, work well. The primers should be similar in length and composition, so that their predicted melting temperatures (T,, the temperature at which 50% of the strands are separated) are within 5°C. From Methods in Molecular Biology, Vol 9 Protocols in Human Molecular GenetIcs Edited by. C. Mathew Copyright Q 1991 The Humana Press Inc., Clifton, NJ 1 2 . . . . . l . . . . . . Highquality reagents are necessary for efficient amplification: particu- larly important are the DNA polymerase -usually the heat-stable enzyme from the thermophilic bacterium Thus aquaticus (e.g., Per-kin-Elmer/Cetus AmpliTaq@)-and the deoxynucleoside triphosphates (dNTPs). Stocks can be prepared as follows: 1. lMKC1, 100 mL. 2. lMTris-HCl, pH 8.3 at 25”C, 100 mL. Bangham GC content should be similar to the GC content of the template and of the other primer, ideally 5040% GC. Binding site on target DNA: conserved region of sequence, ending on a nondegenerate base, e.g., first or second base of a conserved amino acid. No selfcomplementarity (to avoid secondary structures) or complement- arity with the other primer. Computer programs are available to help identify such complementarity. No runs of three or more Gs or Cs at the 3’ end of the primer. If mismatches between primer and template are known or likely to occur, these should be minimized at the 3’ end of the primer, i.e., where the DNA polymerase binds. Highly degenerate primers may work under nonstringent reaction conditions, provided that at least three bases match at the 3’ end of the primer (4). Restriction sites can be included in the primer to help in efficient and directional cloning of the amplified product. The ideal target sequence (template) to be amplified has the following features: Length: 150300 bp. Lengths between 100 and 2000 bp can, however, often be amplified efficiently. Unique sequence, to avoid competition from unwanted templates. High copy number, to minimize the number of cycles of amplification required. PCR is, of course, highly efficient in detecting rare DNA spe- cies, but the risk of confusion with low-abundance contaminating DNA species increases if the target copy number is low. A diagnostic restriction enzyme site, to help verify amplification of the correct product. An intron sequence, to distinguish genomic amplification product from those amplified from cDNA or contaminating DNA (see Section 6). A sequence that can be detected specifically with a probe already in the laboratory. 3. Reagents Getting Started in PCR 3. O.lMMgCl,, 100 mL. 4. 0.2% Gelatin (Difco), 100 mL. 3 Solutions l-4 should be autoclaved and stored in 20-mL aliquots at room temperature. 5. Oligonucleotide primers: 50 @Iupstream primer (300 pg/mL of a 20-mer) ; 50 PA4 downstream primer (300 pg/mL of a 20-mer) . 6. 100 mMdNTPs at neutral pH (e.g., Pharmacia, Central Milton Keynes, Buckinghamshire, UK), stored at -80°C in aliquots of 5 or 10 uL. To minimize the risk of cross-contamination with DNA templates from plasmids or previous amplification reactions, the stock solutions may be irra- diated with UV light at 254 nm, e.g., 10 min in a Stratalinker 1800TM (Stratagene, Cambridge, UK). A 1-mL stock of 2x amplification solution con- taining all components except Tuq polymerase and DNA template can be made, and stored at -20°C in 50-p.L aliquots in siliconized 0.5-mL polypro pylene tubes. The reaction mixture is then completed by adding the DNA template, Taq polymerase (e.g., l-2.5 Cetus U of AmpliTaq@), and sufbcient sterile water to bring the vol to 100 p.L 4. Design of Reaction Mixture For many purposes, the reaction mixture given in Table 1 will give effi- cient and specific amplification. However, there are a few variables that criti- cally affect the efficiency and specificity of the reaction; the most important of these are the magnesium ion concentration and the oligonucleotide primer concentration (see below and Section 6). The optimal number of DNA molecules in the template is between 105 and lo6 (3). For single-copy genes, this corresponds to approx 1 p.g of human genomic DNA and 1 pg of a 6kbp plasmid. Optimization of the reaction mixture for a particular pair of oligo nucleotide primers frequently involves two further steps: 1. Optimize Mg2+ concentration. Amplify the template with the following concentrations of Mg 2+: 1.5 (asabove); 3.0; 4.5; 6.0; and 7.5 mM. Certain primer pairs may require further, finer adjustment of Mg2+ concentra- tion, to within 0.5 mM. 2. Optimize primer concentrations. Amplify the template with the best Mg2+ concentration (as determined above), with the following concentrations of each primer: 0.05; 0.1; 0.25; 0.5; and 1.0 PM. Certain GGrich templates do not amplify with the above protocol, prob ably because they rapidly adopt stable secondary structures on cooling from 94’C. The addition of dimethyl sulfoxide (DMSO) to the reaction mixture Bangham Table 1 Basic PCR Reaction Mixture Final concentration, Volume, PL, for Reagent in lx 2x buffer, 50 p.L 2x buffer, 1 mL 1MKCl 50 mM 5 100 lMTris-HCl 10 mM 1 20 O.lMMgCI, 1.5 mM 1.5 30 0.2% gelatin 0.01% 5 100 100 mMdGTP 200 nM 0.2 4 100 mM dATP 200 nM 0.2 4 100 mM d’lTP 200 nM 0.2 4 100 mMdCTP 200 nM 0.2 4 50pM5’primer 1ClM 2 40 50 pM 3’ primer lClM 2 40 Sterile, deronized water - 33 654 (final concentrauon, 10%) may allow successful amplification, but this is not recommended in other cases, since it decreases the efficiency of the poly- merase enzyme by about 50% (5). Addition of an overlay of inert mineral oil (about 50 pL) (e.g., paraffin oil BP, British Pharmacoepia) to the reaction mixture minimizes evapora- tion during amplification, and so increases the efficiency and reproducibility of the reaction (6). However, it is not essential: if siliconized 0.5mL tubes are used, the droplets that condense on the walls of the tube rapidly return to the solution. To reduce the number of components in the mixture, and so reduce the risk of DNA contamination, the mineral oil and gelatin, and in some instances the KCl, may be omitted. 5. Choice of Reaction Conditions As with the reaction mixture design (Section 4), the following condi- tions serve to amplify efficiently and specifically in many cases. However, there are frequent instances in which the conditions need to be changed for a particular pair of primers. The most important variable to be optimized for a given primer pair is the annealing temperature. This adjustment is a highly empirical process; for example, the annealing temperature may need to be set at, or even above, the predicted T, of a primer (note that the formula given below for estimating the T, takes no account of the magnesium ion concentration). Getting Started in PCR 5.1. Denuturation (94°C) 5 Incomplete denaturation is a frequent cause of failure of PCR. In the initial denaturation step, we use 5 min for a genomic DNA template and 2 min for a plasmid template. In subsequent cycles, 20-30 s at 94°C is adequate. If much longer times are required for successful amplification, the tempera- ture in the reaction mixture itself should be measured with a thermocouple of low specific heat capacity to verify that the solution actually reaches the temperature required for denaturation. 5.2. Annealing (30-60 s) First calculate the approximate T, of the oligonucleotide primers, using a simple formula (7), such as: T,=2x(AtT)t4x(GtC) in ‘C. Then set the annealing temperature at 5°C below the lower of the two predicted T,s. If nonspecific amplification products are a particular problem, anneal- ing and extension can be performed in a single step at between 60 and i’2”C. 5.3. Extension (72°C) Allow 1 min/l kbp of desired product. If the required product is short (<ZOO bp), then the extension step can be omitted, since Tuq polymerase is sufficiently active at lower temperatures to complete the reaction during the transition between the annealing temperature and the denaturauon temperature. 6. Troubleshooting in PCR It is now widely realized that the remarkable sensitivity of PCR is also its main limitation, because a single contaminating molecule of DNA contain- ing the target sequence may be amplified, leading to potentially serious mis- interpretation of the results (8-11) The standards of cleanliness required in making up the solutions are therefore higher than for almost any other laboratory procedure, albeit for different reasons. The main precautions to be taken to avoid false positive results in PCR are listed in Table 2, in approximate order of importance. It is essential to include in each experiment a tube containing all the components except the DNA template, and to examine the products on a gel stained with ethldium bromide, to look for contamination of the reaction mixture. Bangham Table 2 Precautions to Avoid DNA Contamination of PCR Reactions 1. 2. 3. 4. 5. 6. 7. Make up reaction mixtures in a laboratory in which plasmids containing the target sequence are never handled. Neuertake amphfied product mto this labo- ratory. Many workers make up therr PCR solutions in lammar-flow hoods. Wear gloves when making up solutions; avoid touching the inside of the tube cap. To make up reactron mixtures, use pipets that are never used to handle plas- mid or amplificatton productswtth the appropriate sequence. We recommend “positive displacement” pipets (e.g., G&on “Mrcroman”), wrth ups contaunng disposable plungers that prevent aerosol contact or direct contact between the prpet barrel and the solution. For handling small volumes (0.5-10 uL), calibrated drsposable glass microcapillaries are very useful (e.g , Drummond PCR microprpets) . Ahquot reagents and reaction buffers, and use each ahquot only once. See also Sections 2 and 3. Irradiate solutions used in PCR wrth UV. This was shown to abohsh the amph- fication of plasmid that was dehberately added to PCR mixtures (8) Solutions contaming all components except the DNA template can safely be irradiated for 10 min on a 30.5nm-wavelength laboratory UV transrllummator, without denaturing the primers or the enzyme. Avoid reamplificauon of primary amphfied products, rf possible. If amphfica- tion of gel-punfied DNA fragments is necessary, irradiate the agarose gel and its running buffer in the gel apparatus with 254nm W before running: 10 min in a Stratalinker 1800TM (Stratagene) is sufficient. Some workers find that contammation is abolished only when the person mak- ing up the solutions wears a surgical face mask and someumes a harr net (9, J. Todd, personal communication). In some cases (for example, in RNAviruses), it may be possible to arnpli- fy between conserved nucleotide sequences, across a highly variable sequence. If the frequency of nucleotide differences between two amplified products greatly exceeds the error rate of Tuq polymerase, then DNA crosscontamin- ation can be excluded beyond reasonable doubt (II, 12). The dose of W radiation required to prevent amplification depends on the size and the base composition of the potential contaminating species (13). Ideally, the dose should be titrated with a given template and a known contaminant with the W source used in the laboratory. The other common problems in PCR relate to the specificity and effr- ciency of amplification of the required product, avoiding amplification from partial matches between the primers and template. Some of these have been addressed above (seeSections 3 and.5); asummaryof the most frequent causes and their remedies is given in Table 3. Getting Started in PCR Table 3 Troubleshooting in PCR 7 Problem Causes Remedies No detectable product after repeated at- tempts Multiple bands on agarose gel of ampli- fied product Continuous “smear” of amplified product on agarose gel Predominance of very high mol wt amplified product “Primer dimer’% Inadequate melting of DNA template Target sequence too rare Annealing temperature too high CC content of target sequence too high Primers anneal to each other (primer dimer) or to themselves “Overamplificauon” Primers too short or degenerate Concentration of dNTPs or of enzyme too high Annealing temperature too low for CC content of primers “Overamplification” Reamplification of primary amplified product Complementanty between 3’ ends of pnmers Increase ume in denature step Increase number of cycles (up to 60) Lower temperature by 5°C Try 10% DMSO in reaction See note a Reduce number of cycles; reduce exten- sron time See no& a Reduce either by 2-10x Raise annealing tempera- ture by 5°C Reduce number of cycles Gel-purify primary product before reamplifcauon See note a p In each case, the remedy 1s to increase the stringency of the reacuon by increasing the annealing temperature orreducing the primer concentratton orboth b “Overamplification” denotes the use of too many cycles of PCR, which favors the amphficauon of mismatched or nonspectfic DNA products. For amplification of a smgle- copy gene from genomic DNA, 35 cycles should be enough, but more cycles may be needed for a rare species, such as a low-copy-number mfectious agent c The “primer dimer” results from annealing and polymerization of the 5’ pnmer on the z)’ pnmer, and appears as a fuzzy low-molwt band on an ethtdmm bromtde stamed agarose gel 8 Bangham If the problem is one of persistent failure to amplify any band, it may be necessary to choose a different sequence for one or both primers: certain sequences are very inefficient as PCR primers, for unknown reasons. If this is suspected, each primer should be tested in a PCR reaction with another PCR primer of demonstrated efficacy, from the same template sequence (if avail- able). In this way it is frequently possible to show which of the two primers is at fault. 1. 6. 7. 8. 9. 10. 11. 12. 13. References Saiki, R., Scharf, S., Faloona, F., Mullis, K. B., Horn, G T , Erhch, H A , and Amhelm, N (1985) Enzymatic amplification of betaglobin genomrc sequences and restriction analysis for diagnosis of sickle cell anemia. Snence 230, 1350-1354 Erhch, H. A., ed. (1989) PCR Technology: f+wu+!es and Aj$dwatzon f&r DNA Amplzfica- hon. Stockton, New York. Innis, M. A., Gelfand, D. H., Snmsky, J. J., and White, T. J , eds (1990) PCR Protocols. A Gurde to Methods and App1rcat:on.s. Academic, New York. Sommer, R. and Tautz, D. (1989) Muumal homology requirements for PCR primers Nuckic Ands Res. 17,6’749. Gelfand, D. H. and Whtte, T. J. (1990) Thermostable DNA polymerases, m PCR Protc- cols: A Guade to Methods and Apphcatzonr Inms, M A , Gelfand, D H , Snmsky, J J , and White, T. J., eds. Academic, New York, p. 129 Mezei, L. M. (1990) Effect of oil overlay on PCR amphficauon, m Amp2ajicatron.s Perkm- Elmer, Norwalk, CT, vol. 4, p. 11. Them, S. L. and Wallace, R. B. (1986) The use of synthetic ohgonucleoudes as spe- cific hybndtzation probes m the diagnosis of genetic disorders, m Human Gen&c Dzs- eases: A fiactrcal Ap@ach. K. E. Davies, ed. IRL, Oxford, UK, pp. 33-50 Sarkar G. and Sommer, S. S. (1990) Shedding light on PCR contammauon. Nature 343,27. Kitchin, P. A., Szotyori, Z., Fromholc, C , and Almond, N (1990) Avoidance of false positives. Natun 344,201. Kwok, S. and Higuchi, R. (1989) Avordmg false positives wnh PCR Nature339,237,238 Bangham, C. R. M , Nightingale, S., Cruickshank, J K., and Daenke, S. (1989) PCR analysis of DNA from muluple sclerosis patients for the presence of HTLV-I Sczence 246,821. Daenke, S., Nightingale, S., Crurckshank, J. K, and Bangham, C R M (1990) Se- quence vanants of human T-cell lymphotropic virus type I from patients with tropical spasuc paraparesrs and adult T-cell leukemia do not distmgursh neurological from leukemic isolates. j. Viral. 64,12%-l 282 Crmmo, G. D., Metchette, K., Isaacs, S. T., and Zhu, Y. S. (1990) More false positive problems. Nature 345, ‘7’73,174. CHAFTER 2 Direct DNA Sequencing of Complementary DNAAmplified by the Polymerase Chain Reaction Richard A. Gibbs, Phi-Nga Nmyen, and C. Thomas Caskey 1. Introduction Protocols for the sequence analysis of conventional single-stranded or double-stranded DNA templates are often unsuitable for the direct sequenc- ing of DNA fragments generated by the polymerase chain reaction (PCR) (1,2). The features that can distinguish PCR products as templates for se- quencing include (a) contamination of the reactions by nonspecific PCR amplification products that are complementary to the sequencing primer, (b) the persistence of “leftover” PCR primers from the amplification reac- tions, and (c) the potential for competition between one strand of the ampli- fied fragment and the oligonucleotide used for the sequencing. The various approaches that have been used to overcome these problems include 1. The use of 5’-end-labeled DNA-sequencing primers that are comple- mentary to regions between the PCR primers (3); 2. Gel purification of amplified DNA to remove unwanted fragments and primer (4); 3. Spin columns for the separation of leftover primers from high mol wt material (5, 6); 4. “Asymmetric” or knbalanced” PCR priming to generate an excess of single strands during the initial amplification (7); From* Methods in Molecular Bology, Vol. 9. Protocols in Human Molecular GenetIcs Edited by C. Mathew Copyright 0 1991 The I-hJmana Press Inc., Cl&on, NJ 9 10 Gibbs, Nguyen, and Caskey 5. Addition of dimethylsulfoxide (DMSO) to sequencing reactions with short annealing times (8); and 6. The use of several short, high-temperature, sequencing cycles (9). In developing the protocol that is described here (summarized in Fig. l), we have endeavored to avoid the tedious steps of gel purification or col- umn chromatography. Instead, we have developed a twostep reaction proce- dure for template preparation that first allows amplification of a specific fragment and then the production of an excess of one strand. This method is essentially a modification of the asymmetric priming protocol of Gyllensten and Erlich (‘7). The current method can be performed comfortably in two days and enables the reliable generation of DNA sequence ladders that can be resolved as far as the gel system that is used will allow. The technique has been applied for the analysis of transcribed human sequences, for which it is preceded by a reverse transcription reaction. Equal success has been obtained in the analysis of human gene sequences using lo-100 ng of genomic DNA as starting material and there is no reason that virtually any DNA fragment that can be successfully amplified by PCR would not be amenable to this analysis. Features that are modifications of other protocols or that we regard as par- ticularly important are further discussed below. 1. 2. 3. 4. 5. 6. 7. a. 2. Materials (see Note 1) Ribonuclease inhibitor (RNasin; Pharmacia, catalog no. 27-0815-01; 27,000 U/mL). Random hexamer primers (pd(N)s, Pharmacia, catalog no. 27-2166-01; at 5 mg/mL) . 5x POL buffer (250 mMTrisHC1, pH 8.3 at 3’7”C, 40 mM MgCl,, 150 mMKC1; 50 mMdithiothreito1 [D’IT]). Deoxyribonucleotide triphosphate mixture (dNTPs; mixture of 2.5 mM each of dATP, dTTP, dCTP, and dGTP) . Reverse Transcriptase (M-MuLV, Pharmacia catalog no. 27-0925 02;12,000 U/mL). Modified T7 DNA polymerase (SequenaseTM; usually supplied at 12.5 U&L from United States Biochemicals [USB] catalog no. 70722). Dideoxynucleotide terminator mixtures; these are 80~8 pMdeoxy:dideoxy mixtures. The solutions supplied by USB, catalog nos. 70714 (“A” mix), 70716 (“C” mix), 70718 (“G” mix), and 70720 (,T” mix) are appropri- ate. The solutions are thawed and stored in 20-PL aliquots. SequenaseTM (1 .O l.tL) is added to each just before use. Sequencing stop solution (STOP; 95% formamide, 20mMEDTA, 0.05% bromophenol blue, 0.05% xylene cyanol). [...]... product using oligimer #863 The faster-moving bands represent the single-stranded fragments ration for the SSPRs,except when we find that a particular primer set functions much better in the DMSO-containing buffer In that case, the DMSO-containing buffer is used in the SSPR,but great care is taken to avoid salt or protein coprecipitation 9 Sequencing primers: The DNA-sequencing primers are routinely constructed... and kept at -2OOC) 6 Vortex and spin for 15 s Repeat steps 4-6 twice 7 After removing the supernatant, respin the pellet for 15 s and remove residual liquid (including any remaining paraffin) with a drawn-out Pasteur pipet 8 Add 5 ltL of TE to the pellet and resuspend with the automatic plpet Incubate for 5 min at 55OC (either in a water bath or a PCR machine) 9 Spin for 30 s Transfer the supernatan... required The simplest of these include increasing the amount of Tuq DNA polymerase in the APCR mix, titrating the Mg2+ concentration in the PCR buffer, increasingor decreasing the annealing temperature, and increasing the denaturation (94OC) and extension (72OC) times 3 As has been discussed elsewhere (10), the high-throughput APCR template-preparation scheme with recombinant Ml3 clones requires some... and single-strand production: A key step in the analysis is the generation of a single strand by asymmetric priming in a PCR-like reaction As described in the original report of the method, a single PCR is performed with different amounts of each primer (7) Initially there is an exponential increase in the amount of the desired fragment, and then, as one primer is exhausted, the second primer continues... of fluorescent-labeling chemistrres: (a) reporter group at the 5’ terminus of the sequencing primer, and (b) reporter group at the 2’ carbon of the dideoxynucleotide Since the end-labeled fluorescent From Methods Edited by in Molecular C Mathew Bdogy, Copyright Vol 9 Q 1991 29 Protocols m Human The Humana Press Molecular Genetrcs Inc , Clifton, NJ 30 Wilson Reverse primer G-40) in excess (50 pmoles)... 30 s), and annealing (37-65’C, 30 s) The optimum annealing temperature must be determined empirically In initial reactions, allow at least 1 mm of extension/500 bases 13 Direct Sequencing of DNA 5 The final incubation at 68°C is extended 6 Remove the sample from under the oil for 7 min 3.3 Single-Strand-Producing Reactions (see Notes 538) (SSPRs) 1 Take 1 yL of the PCR product to initiate a second... molecules where every possible length is represented in sufficient amounts to be detected by autoradiography after fractionation on a denaturing polyacrylamide gel This results in a “ladder” of bands across four tracks that are read From: Methods in Molecular Edlted by- C Mathew Biology, Vol 9 Protocols m Human Molecular Genetics Copyright Q 1991 The Humana Press Inc , Clifton, NJ 21 22 Green and Giannelli... well-forming combs supplied by Applied Biosystems Detailed instructions for preparing, setting up, and prerunning the gel are provided in the user’s manual 2 DNA-sequencing reactions should be performed in 0.6mL microcentrifuge tubes or, more conveniently, in 96well V- or U-bottom microtiter plates (see Note 5) Care should be taken to keep the reactions away from fluorescent lighting Set up four annealing... at -70°C (or overnight at 4*C), and spin for 30 min in a microfuge Repeat the ammonium acetate precipitation Wash with 70% ethanol, again with 100% ethanol, and dry to completion under vacuum Dis solve the pellet in 10 ltL of Hz0 immediately before use in the DNAsequencing reaction 3.4 Radiolabeling the DNA Sequencing (see Note 9) Primer 1 Kinase reactions contain 20-50 pm01 of primer, 50-70 l.tCi of... spinner to spin down the drops in a microtiter plate Alternatively, tap on the bench to knock the droplets down to the bottom 9 Put tape around the edge of plate and float on 37°C water bath for 5 min 10 Add 2 l.tL of chase solution to every well, spin to mix, and again incubate at 37OC for 5 min 11 Add 2 yL of running dyes to each well, and spin to mix 12 When the gel is ready for loading (see below), . 6. Troubleshooting in PCR It is now widely realized that the remarkable sensitivity of PCR is also its main limitation, because a single contaminating molecule of DNA contain- ing the target. main precautions to be taken to avoid false positive results in PCR are listed in Table 2, in approximate order of importance. It is essential to include in each experiment a tube containing. amplification (7); From* Methods in Molecular Bology, Vol. 9. Protocols in Human Molecular GenetIcs Edited by C. Mathew Copyright 0 1991 The I-hJmana Press Inc., Cl&on, NJ 9 10 Gibbs,