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The Polymerase Chain Reaction Organized by: Dr. Abir Adel SAAD Biotechnology Dept, IGSR, University of Alexandria 1 The Polymerase Chain Reaction (PCR) 12345678910- Principle of the PCR PCR primer design and reaction optimisation Standard PCR protocol Reverse transcription PCR: RNA -> lots of DNA Calculating concentrations for PCR Troubleshooting The applications of PCR PCR Glossary Real time PCR Principle of Sequencing DNA amplification is the cornerstone of modern biotechnology and it is also a key procedure in numerous basic studies involving DNA and other biomolecules. All methods for DNA amplification have rested on the concept of DNA strand complementarity discovered by Watson and Crick fifty years ago. Polymerase chain reaction (PCR) is still the most popular amplification method, however alternatives to PCR have successfully invaded the area. The emergence of such methodologies has significantly widened the range of approaches for DNA amplification and dramatically improved the technological abilities of basic and applied researchers in various fields of life sciences. It will not be an exaggeration to say that now no research related to DNA can be performed without the employment of DNA amplification procedures. Newly-developed protocols including real-time PCR and other new PCR developments, plus several powerful non-PCR isothermal DNA amplification techniques are now being used in the field of molecular biology. 1- Principle of the PCR The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of copies of a gene. This is necessary to have enough starting template for sequencing. The cycling reactions : There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler “Thermocycler”, which can heat and cool the tubes with the reaction mixture in a very short time. 1. Denaturation at 94°C : During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example: the extension from a previous cycle). 2. Annealing at 54°C : The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there 2 are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore. 3. Extension at 72°C : This is the ideal working temperature for the polymerase. The primers, where there are a few bases built in, already have a stronger ionic attraction to the template than the forces breaking these attractions. Primers that are on positions with no exact match, get loose again (because of the higher temperature) and don't give an extension of the fragment. The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template) The different steps in PCR. Because both strands are copied during PCR, there is an exponential increase of the number of copies of the gene. Suppose there is only one copy of the wanted gene before the cycling starts, after one cycle, there will be 2 copies, after two cycles, there will be 4 copies, three cycles will result in 8 copies and so on. 3 The exponential amplification of the gene in PCR. Is there a gene copied during PCR and is it the right size ? Before the PCR product is used in further applications, it has to be checked if : 1. There is a product formed. Though biochemistry is an exact science, not every PCR is successful. There is for example a possibility that the quality of the DNA is poor, that one of the primers doesn't fit, or that there is too much starting template 2. The product is of the right size It is possible that there is a product, for example a band of 500 bases, but the expected gene should be 1800 bases long. In that case, one of the primers probably fits on a part of the gene closer to the other primer. It is also possible that both primers fit on a totally different gene. 3. Only one band is formed. As in the description above, it is possible that the primers fit on the desired locations, and also on other locations. In that case, you can have different bands in one lane on a gel. Verification of the PCR product on gel. The ladder is a mixture of fragments with known size to compare with the PCR fragments. Notice that the distance between the different fragments of the ladder is logarithmic. Lane 1: PCR fragment is approximately 1850 bases long. Lane 2 and 4: the fragments are approximately 800 bases long. Lane 3: no product is formed, so the PCR failed. Lane 5: multiple bands are formed because one of the primers fits on different places. 4 2- PCR PRIMER DESIGN AND REACTION OPTIMISATION Contents 1234567891011121314- Factors Affecting the PCR Denaturing Temperature and Time Annealing Temperature and Primer Design Primer Length Degenerate Primers Elongation Temperature and Time Reaction Buffer Cycle Number Nested Primer PCR Labelling of PCR products with digoxygenin-11-dUTP Helix Destabilisers / Additives Useful Universal cDNA PCR Primer A simple set of rules for primer sequence design REFERENCES Factors Affecting the PCR: Denaturing Temperature and time The specific complementary association due to hydrogen bonding of singlestranded nucleic acids is referred to as "annealing": two complementary sequences will form hydrogen bonds between their complementary bases (G to C, and A to T or U) and form a stable double-stranded, anti-parallel "hybrid" molecule. One may make nucleic acid (NA) single-stranded for the purpose of annealing - if it is not singlestranded already, like most RNA viruses - by heating it to a point above the "melting temperature" of the double- or partially-double-stranded form, and then flash-cooling it: this ensures the "denatured" or separated strands do not re-anneal. Additionally, if the NA is heated in buffers of ionic strength lower than 150mM NaCl, the melting temperature is generally less than 100oC - which is why PCR works with denaturing temperatures of 91-97oC. Taq polymerase is given as having a half-life of 30 min at 95oC, which is partly why one should not do more than about 30 amplification cycles: however, it is possible to reduce the denaturation temperature after about 10 rounds of amplification, as the mean length of target DNA is decreased: for templates of 300bp or less, denaturation temperature may be reduced to as low as 88oC for 50% (G+C) templates (Yap and McGee, 1991), which means one may do as many as 40 cycles without much decrease in enzyme efficiency. "Time at temperature" is the main reason for denaturation / loss of activity of Taq: thus, if one reduces this, one will increase the number of cycles that are possible, whether the temperature is reduced or not. Normally the denaturation time is 1 min at 94oC: it is possible, for short template sequences, to reduce this to 30 sec or less. 5 Increase in denaturation temperature and decrease in time may also work: Innis and Gelfand (1990) recommend 96oC for 15 sec. Annealing Temperature and Primer Design Primer length and sequence are of critical importance in designing the parameters of a successful amplification: the melting temperature of a NA duplex increases both with its length, and with increasing (G+C) content: a simple formula for calculation of the Tm is Tm = 4(G + C) + 2(A + T)oC. Thus, the annealing temperature chosen for a PCR depends directly on length and composition of the primer(s). One should aim at using an annealing temperature (Ta) about 5oC below the lowest Tm of the pair of primers to be used (Innis and Gelfand, 1990). A more rigorous treatment of Ta is given by Rychlik et al. (1990): they maintain that if the Ta is increased by 1oC every other cycle, specificity of amplification and yield of products <1kb in length are both increased. One consequence of having too low a Ta is that one or both primers will anneal to sequences other than the true target, as internal single-base mismatches or partial annealing may be tolerated: this is fine if one wishes to amplify similar or related targets; however, it can lead to "non-specific" amplification and consequent reduction in yield of the desired product, if the 3'-most base is paired with a target. A consequence of too high a Ta is that too little product will be made, as the likelihood of primer annealing is reduced; another and important consideration is that a pair of primers with very different Ta may never give appreciable yields of a unique product, and may also result in inadvertent "asymmetric" or single-strand amplification of the most efficiently primed product strand. Annealing does not take long: most primers will anneal efficiently in 30 sec or less, unless the Ta is too close to the Tm, or unless they are unusually long. An illustration of the effect of annealing temperature on the specificity and on the yield of amplification of Human papillomavirus type 16 (HPV-16) is given below (Williamson and Rybicki, 1991: J Med Virol 33: 165-171). Plasmid and biopsy sample DNA templates were amplified at different annealing temperatures as shown: note that while plasmid is amplified from 37 to 55oC, HPV DNA is only specifically amplified at 50oC. 6 Primer Length The optimum length of a primer depends upon its (A+T) content, and the Tm of its partner if one runs the risk of having problems such as described above. Apart from the Tm, a prime consideration is that the primers should be complex enough so that the likelihood of annealing to sequences other than the chosen target is very low. For example, there is a 1⁄4 chance (4-1) of finding an A, G, C or T in any given DNA sequence; there is a 1/16 chance (4-2) of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence. Thus, a sixteen base sequence will statistically be present only once in every 416 bases (=4 294 967 296, or 4 billion): this is about the size of the human or maize genome, and 1000x greater than the genome size of E. coli. Thus, the association of a greater-than-17-base oligonucleotide with its target sequence is an extremely sequence-specific process, far more so than the specificity of monoclonal antibodies in binding to specific antigenic determinants. Consequently, 17-mer or longer primers are routinely used for amplification from genomic DNA of animals and plants. Too long a primer length may mean that even high annealing temperatures are not enough to prevent mismatch pairing and nonspecific priming. Degenerate Primers For amplification of cognate sequences from different organisms, or for "evolutionary PCR", one may increase the chances of getting product by designing "degenerate" primers: these would in fact be a set of primers which have a number of options at several positions in the sequence so as to allow annealing to and amplification of a variety of related sequences. For example, Compton (1990) describes using 14-mer primer sets with 4 and 5 degeneracies as forward and reverse primers, respectively, for the amplification of glycoprotein B (gB) from related herpesviruses. The reverse primer sequence was as follows: TCGAATTCNCCYAAYTGNCCNT where Y = T + C, and N = A + G + C + T, and the 8-base 5'-terminal extension comprises a EcoRI site (underlined) and flanking spacer to ensure the restriction enzyme can cut the product (the New England Biolabs catalogue gives a good list of which enzymes require how long a flanking sequence in order to cut stub ends). Degeneracies obviously reduce the specificity of the primer(s), meaning mismatch opportunities are greater, and background noise increases; also, increased degeneracy means concentration of the individual primers decreases; thus, greater than 512-fold degeneracy should be avoided. However, I have used primers with as high as 256- and 1024-fold degeneracy for the successful amplification and subsequent direct sequencing of a wide range of Mastreviruses against a background of maize genomic DNA (Rybicki and Hughes, 1990). 7 Primer sequences were derived from multiple sequence alignments; the mismatch positions were used as 4-base degeneracies for the primers (shown as stars; 5 in F and 4 in R), as shown above. Despite their degeneracy, the primers could be used to amplify a 250 bp sequence from viruses differing in sequence by as much as 50% over the target sequence, and 60% overall. They could also be used to very sensitively detect the presence of Maize streak virus DNA against a background of maize genomic DNA, at dilutions as low as 1/109 infected sap / healthy sap (see below). Some groups use deoxyinosine (dI) at degenerate positions rather than use mixed oligos: this base-pairs with any other base, effectively giving a four-fold degeneracy at any postion in the oligo where it is present. This lessens problems to do with depletion of specific single oligos in a highly degenerate mixture, but may result in too high a degeneracy where there are 4 or more dIs in an oligo. 8 Elongation Temperature and Time This is normally 70 - 72oC, for 0.5 - 3 min. Taq actually has a specific activity at 37oC which is very close to that of the Klenow fragment of E coli DNA polymerase I, which accounts for the apparent paradox which results when one tries to understand how primers which anneal at an optimum temperature can then be elongated at a considerably higher temperature - the answer is that elongation occurs from the moment of annealing, even if this is transient, which results in considerably greater stability. At around 70oC the activity is optimal, and primer extension occurs at up to 100 bases/sec. About 1 min is sufficient for reliable amplification of 2kb sequences (Innis and Gelfand, 1990). Longer products require longer times: 3 min is a good bet for 3kb and longer products. Longer times may also be helpful in later cycles when product concentration exceeds enzyme concentration (>1nM), and when dNTP and / or primer depletion may become limiting. Reaction Buffer Recommended buffers generally contain : * * * * * * v/v) 10-50mM Tris-HCl pH 8.3, up to 50mM KCl, 1.5mM or higher MgCl2, primers 0.2 - 1uM each primer, 50 - 200uM each dNTP, gelatin or BSA to 100ug/ml, and/or non-ionic detergents such as Tween-20 or Triton X-100 (0.05 - 0.10% PCR is supposed to work well in reverse transcriptase buffer, and vice-versa, meaning 1-tube protocols (with cDNA synthesis and subsequent PCR) are possible (Krawetz et al., 19xx; Fuqua et al., 1990). Higher than 50mM KCl or NaCl inhibits Taq, but some is necessary to facilitate primer annealing. [Mg2+] affects primer annealing; Tm of template, product and primer-template associations; product specificity; enzyme activity and fidelity. Taq requires free Mg2+, so allowances should be made for dNTPs, primers and template, all of which chelate and sequester the cation; of these, dNTPs are the most concentrated, so [Mg2+] should be 0.5 - 2.5mM greater than [dNTP]. A titration should be performed with varying [Mg2+] with all new template-primer combinations, as these can differ markedly in their requirements, even under the same conditions of concentrations and cycling times/temperatures. Some enzymes do not need added protein, others are dependent on it. Some enzymes work markedly better in the presence of detergent, probably because it prevents the natural tendency of the enzyme to aggregate. Primer concentrations should not go above 1uM unless there is a high degree of degeneracy; 0.2uM is sufficient for homologous primers. Nucleotide concentration need not be above 50uM each: long products may require more, however. 9 Cycle Number The number of amplification cycles necessary to produce a band visible on a gel depends largely on the starting concentration of the target DNA: Innis and Gelfand (1990) recommend from 40 - 45 cycles to amplify 50 target molecules, and 25 - 30 to amplify 3x105 molecules to the same concentration. This non-proportionality is due to a so-called plateau effect, which is the attenuation in the exponential rate of product accumulation in late stages of a PCR, when product reaches 0.3 - 1.0 nM. This may be caused by degradation of reactants (dNTPs, enzyme); reactant depletion (primers, dNTPs - former a problem with short products, latter for long products); end-product inhibition (pyrophosphate formation); competition for reactants by non-specific products; competition for primer binding by re-annealing of concentrated (10nM) product (Innis and Gelfand, 1990). If desired product is not made in 30 cycles, take a small sample (1ul) of the amplified mix and re-amplify 20-30x in a new reaction mix rather than extending the run to more cycles: in some cases where template concentration is limiting, this can give good product where extension of cycling to 40x or more does not. A variant of this is nested primer PCR: PCR amplification is performed with one set of primers, then some product is taken - with or without removal of reagents - for reamplification with an internally-situated, "nested" set of primers. This process adds another level of specificity, meaning that all products non-specifically amplified in the first round will not be amplified in the second. This is illustrated below: 10 This gel photo shows the effect of nested PCR amplification on the detectability of Chicken anaemia virus (CAV) DNA in a dilution series: the PCR1 just detects 1000 template molecules; PCR2 amplifies 1 template molecule (Soiné C, Watson SK, Rybicki EP, Lucio B, Nordgren RM, Parrish CR, Schat KA (1993) Avian Dis 37: 467-476). Labelling of PCR products with digoxygenin-11-dUTP (DIG; Roche) need be done only in 50uM each dNTP, with the dTTP substituted to 35% with DIG-11-dUTP. NOTE: that the product will have a higher MW than the native product! This results in a very well labelled probe which can be extensively reused, for periods up to 3 years. Helix Destabilisers / Additives With NAs of high (G+C) content, it may be necessary to use harsher denaturation conditions. For example, one may incorporate up to 10% (w or v/v) : * dimethyl sulphoxide (DMSO), * dimethyl formamide (DMF), * urea * or formamide in the reaction mix: these additives are presumed to lower the Tm of the target NA, although DMSO at 10% and higher is known to decrease the activity of Taq by up to 50% (Innis and Gelfand, 1990; Gelfand and White, 1990). Additives may also be necessary in the amplification of long target sequences: DMSO often helps in amplifying products of >1kb. Formamide can apparently dramatically improve the specificity of PCR (Sarkar et al., 1990), while glycerol improves the amplification of high (G+C) templates (Smith et al., 1990). Polyethylene glycol (PEG) may be a useful additive when DNA template concentration is very low: it promotes macromolecular association by solvent exclusion, meaning the pol can find the DNA. cDNA PCR A very useful primer for cDNA synthesis and cDNA PCR comes from a sequencing strategy described by Thweatt et al. (1990): this utilised a mixture of three 21-mer primers consisting of 20 T residues with 3'-terminal A, G or C, respectively, to 11 sequence inside the poly(A) region of cDNA clones of mRNA from eukaryotic origin. I have used it to amplify discrete bands from a variety of poly(A)+ virus RNAs, with only a single specific degenerate primer upstream: the T-primer may anneal anywhere in the poly(A) region, but only molecules which anneal at the beginning of the poly(A) tail, and whose 3'-most base is complementary to the base next to the beginning of the tail, will be extended. eg: 5'-TTTTTTTTTTTTTTTTTTTTTTTTT(A,G,C)-3' works for amplification of Potyvirus RNA, and eukaryotic mRNA A simple set of rules for primer sequence design is as follows (adapted from Innis and Gelfand, 1991): 1. primers should be 17-28 bases in length; 2. base composition should be 50-60% (G+C); 3. primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming; 4. Tms between 55-80oC are preferred; 5. runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided; 6. 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product; 7. primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided. Examples of inter- and intra-primer complementarity which would result in problems: 12 REFERENCES Compton T (1990). Degenerate primers for DNA amplification. pp. 39-45 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York. Fuqua SAW, Fitzgerald SD and McGuire WL (1990). A simple polymerase chain reaction method for detection and cloning of low-abundance transcripts. BioTechniques 9 (2):206-211. Gelfand DH and White TJ (1990). Thermostable DNA polymerases. pp. 129-141 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York. Innis MA and Gelfand DH (1990). Optimization of PCRs. pp. 3-12 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York. Krawetz SA, Pon RT and Dixon GH (1989). Increased efficiency of the Taq polymerase catalysed polymerase chain reaction. Nucleic Acids Research 17 (2):819. Rybicki EP and Hughes FL (1990). Detection and typing of maize streak virus and other distantly related geminiviruses of grasses by polymerase chain reaction amplification of a conserved viral sequence. Journal of General Virology 71:2519-2526. Rychlik W, Spencer WJ and Rhoads RE (1990). Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Research 18 (21):6409-6412. Sarkar G, Kapeiner S and Sommer SS (1990). Formaqmide can drrastically increase the specificity of PCR. Nucleic Acids Research 18 (24):7465. Smith KT, Long CM, Bowman B and Manos MM (1990). Using cosolvents to enhance PCR amplification. Amplifications 9/90 (5):16-17. Thweatt R, Goldstein S and Reis RJS (1990). A universal primer mixture for sequence determination at the 3' ends of cDNAs. Analytical Biochemistry 190:314-316. Wu DY, Ugozzoli L, Pal BK, Qian J, Wallace RB (1991). The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction. DNA and Cell Biology 10 (3):233-238. Yap EPH and McGee JO'D (1991). Short PCR product yields improved by lower denaturation temperatures. Nucleic Acids Research 19 (7):1713. 13 3- Standard PCR Protocol Contents * * * * * * * * * * * * Recommended Reagent Concentrations Recommended Reaction Conditions Initial Conditions Temperature Cycling "Hot Start" PCR Asymmetric PCR for ssDNA Production Detecting Products Labelling PCR Products with Digoxigenin Cleaning PCR Products Sequencing PCR Products Cloning PCR Products AND ALWAYS REMEMBER: Recommended Reagent Concentrations: * * * * Primers: 0.2 - 1.0 uM Nucleotides: 50 - 200 uM EACH dNTP Dimethyl sulphoxide (DMSO): 0 - 10% (v/v) Taq polymerase: 0.5 - 1.0 Units/50ul rxn Target DNA: 1 ng - 1 ug (NB: higher concn for total genomic DNA; lower for plasmid / purified DNA / virus DNA target) Buffer: use proprietary or home-made 10x rxn mix; eg: Cetus, Promega. This should contain: minimum of 1.5mM Mg2+, usually some detergent, perhaps some gelatin or BSA. Promega now supply 25mM MgCl2, to allow user-specified [Mg2+] for reaction optimisation with different combinations of primers and targets. MAKE POOLED MASTER MIX OF REAGENTS IN ABSENCE OF DNA using DNA-free pipette, then dispense to individual tubes (using DNA-free pipette), and add DNA to individual reactions USING PLUGGED TIPS. NOTE: USE PLUGGED PIPETTE TIPS: prevents aerosol contamination of pipettes. Use of detergents is recommended only for Taq from Promega (up to 0.1% v/v, Triton X-100 or Tween-20). DMSO apparently allows better denaturation of longer target sequences (>1kb) and more product. DO NOT USE SAME PIPETTE FOR DISPENSING NUCLEIC ACIDS AS YOU USE FOR DISPENSING REAGENTS Remember sample volume should not exceed 1/10th reaction volume, and sample DNA/NTP/primer concentrations should not be too high as otherwise all available Mg2+ is chelated out of solution and enzyme reactivity is adversely affected. Any increase in dNTPs over 200uM means [Mg2+] should be re-optimised. 14 Low primer, target, Taq, and nucleotide concentrations are to be favoured as these generally ensure cleaner product and lower background, perhaps at the cost of detection sensitivity. Recommended Reaction Conditions: Initial Conditions: Initial denaturation at start: 92 - 97oC for 3 - 5 min. If you denature at 97oC, denature sample only; add rest of mix after reaction colls to annealing temperature (prevents premature denaturation of enzyme). Initial annealing temperature: as high as feasible for 3 min (eg: 50 - 75oC). Stringent initial conditions mean less non-specific product, especially when amplifying from eukaryotic genomic DNA. Initial elongation temperature: 72oC for 3 - 5 min. This allows complete elongation of product on rare templates. Temperature Cycling: * * * * * 92 - 94oC for 30 - 60 sec (denature) 37 - 72oC for 30 - 60 sec (anneal) 72oC for 30 - 60 sec (elongate) (60 sec per kb target sequence length) 25 - 35 cycles only (otherwise enzyme decay causes artifacts) 72oC for 5 min at end to allow complete elongation of all product DNA NOTE: "Quickie" PCR is quite feasible: eg, [94oC 30 sec / 45oC 30 sec / 72oC 30 sec] x 30, for short products (200 - 500 bp). DON'T RUN TOO MANY CYCLES: if you don't see a band with 30 cycles you probably won't after 40; rather take an aliquot from the reaction mix and re-PCR with fresh reagents. "Hot Start" PCR: In certain circumstances one wishes to avoid mixing primers and target DNA at low temperatures in the presence of Taq polymerase: Taq pol is almost as efficient as Klenow pol at 37oC; consequently, if primers mis-anneal at low temperature prior to initial template denaturation, "non-specific" amplification may occur. This may be avoided by only adding enzyme after the initial denaturation, before the reaction cools to the chosen annealing temperature. This is most conveniently done by putting wax "gems"TM into the reaction tube after addition of everything except enzyme, then putting enzyme on top of the gem: the wax melts when the temperature reaches +/80oC, and the enzyme mixes with the rest of the reaction mix while the molten wax floats on top and seals the mix, taking the place of mineral oil. Information is that "gems" may be substituted by VaselineTM. 15 Asymmetric PCR for ssDNA Production: Simply use a 100:1 molar ratio of the two primers (eg: primer 1 at 0.5uM, primer 2 at 0.005uM). This allows production of mainly ssDNA of the sense of the more abundant primer, which is useful for sequencing purposes or making ssDNA probes. Detecting Products: Take 1/10th - 1/3rd of the reaction mix CAREFULLY from under the oil or from under the Vaseline or solidified wax, using a micropipette with plugged tip, IN AN AREA AWAY FROM YOUR PCR PREPARATION AREA! Mix this with some gel loading buffer(1:1 - 1:5 mix:loading buffer): this is TBE containing 10 - 20% glycerol or sucrose and a dash of bromophenol blue (BPB) tracking dye. Load 5 - 30ul of sample into wells of 0.8 - 3.0% submarine agarose gel made up in TBE, preferably containing 50ng/ml ethidium bromide. Run at 80 -120 volts (not too slow or small products diffuse; not too fast or bands smear) until BPB reaches end of gel (large products) or 2/3 down gel (small products). Use DNA markers going from 2kb down to 100 bp or less (recommend BM PCR markers). View on UV light box at 254 - 300 nm, photo 1 - 5 sec. NOTE: Small products are best seen on 3% agarose gels that have been run fast (eg: 100 volts), with BPB run to 1⁄2 - 2/3 down the gel. It is best to include EthBr in the gel AND in the gel buffer, as post-electrophoresis staining can result in band smearing due to diffusion, and if there is no EthBr in the buffer the dye runs backwards out of the gel, and smaller bands are stripped of dye and are not visible. NUSIEVE TM gel (FMC Corp) can also be used for small products - better resolution than agarose. Polyacrylamide gels can be silver stained by simple protocols for extreme sensitivity of detection. Gels can be blotted directly after soaking in 0.5M NaOH / 1.5M NaCl for 10-20 min: "dry blotting" works well (eg: gel is over- and under-layered with paper towel stacks and pressed; bands transfer up and down), as does classic "Southern" blotting. Bands blotted in this way are already covalently fixed onto nylon membranes, and simply need a rinse in 5xSSPE before prehybridisation. 16 The example shown is of detection of Human papillomavirus type 16 (HPV-16) DNA amplified from cervical biopsy samples (Williamson A-L, Rybicki EP (1991) Detection of genital human papillomaviruses by polymerase chain reaction amplification with degenerate nested primers. J Med Virol 33: 165-171). The left panel is a photo of an EthBR-stained 2% agarose gel; the right is an autoradiograph of a Southern blot probed with 32P-labelled HPV-16 DNA. Note how much more sensitive blotting is, and how much more specific the detection is. Labelling PCR Products with Digoxigenin PCR products may be very conveniently labelled with digoxigenin-11-dUTP (Boehringer-Mannheim) by incorporating the reagent to 10-35% final effective dTTP concentration in a nucleotide mix of final concentration 50-100uM dNTPs (Emanual, 1991; Nucleic Acids Res 19: 2790). This allows substitution to a known extent of probes of exactly defined length, which in turn allows exactly defined bybridisation conditions. It is also the most effective means of labelling PCR products, as it is potentially unsafe and VERY expensive to attempt to do similarly with 32P-dNTPs, and nick-translation or random primed label incorporation are unsuitable because the templates are often too small for efficient labelling. Make a DIG-dNTP mix for PCR as follows: DIG NUCLEOTIDE MIX CONCENTRATIONS * * * * * Dig-11-dUTP 350 uM dTTP 650 uM dATP 1 mM dCTP 1 mM dGTP 1 mM For each 50 ul of probe synthesized, a 1/10 dilution is made of the DIG-nucleotide mix when added to the other reagents as described above. The products may be analyzed by agarose gel electrophoresis - NOTE: PRODUCTS ARE LARGER THAN NON-SUBSTITUTED PRODUCT - and detected directly on blots immunologically. Probes can be used as 5-10 ul aliquots directly from PCR product mixes, mixed with hybridisation mix and denatured. Probes can be re-used up to 10 times, stored frozen in between experiments and boiled to denature. 17 Cleaning PCR Products * Getting rid of mineral oil: simply add 50ul of chloroform to the reaction vial, vortex and centrifuge briefly, and remove the "hanging droplet" of AQUEOUS solution with a micopipette. * Getting rid of wax or Vaseline: simply "spear" wax gem and remove; do as for oil or bottom-puncture tube and blow out aqueous drop for Vaseline. * Cleaning-up DNA: 3 options * a protocol which gives DNA that is clean enough for sequencing is the following: increase volume to 100ul with water, add 10M ammonium acetate soln. to 0.2M final concentration (1/5th volume), add equal volume of isopropanol (propan-2ol), leave on bench 5 min, centrifuge 20 min at 15 000 rpm, remove liquid using pipette, resuspend in 100ul water or TE, repeat precipitation. * Simply do a phenol-CHCl3 extraction (add 20ul phenol to aqueous phase, vortex, add 50ul CHCl3, vortex, centrifuge, remove UPPER aqueous phase, repeat CHCl3 extraction). * Make aqueous phase up to 400ul, and spin through Millipore Ultrafree-MC NMWL 30 000 cartridges (at 6000 rpm in microcentrifuge), wash through with 2x400ul water, collect +/-20ul sample: this is pure enough for sequencing. NOTE: Product is clean enough for restriction digest immediately after reaction; however, phenol-chloroform clean-up is recommended as a minimum. Sequencing PCR Products: This is best done using ssDNA generated by asymmetric PCR, and the "limiting" primer for sequencing. However, efficient sequencing of dsDNA generated by normal PCR is possible using the modification to the SequenaseTM protocol published by Bachmann et al. (1990) (NAR 18: 1309). CLEAN DNA is resuspended in sequencing buffer containing 0.5% Nonidet P-40 and 0.5% Tween-20 and sequencing primer, denatured by heating to 95oC for 5 min, snap-cooled on wet ice, and sequenced by the "close-to-primer" protocol (eg: dilute extension mixes). Cloning PCR Products T-A Cloning Strategy: Taq and other polymerases seem to have a terminal transferase activity which results in the non-templated addition of a single nucleotide to the 3'ends of PCR products. In the presence of all 4 dNTPs, dA is preferentially added; however, use of a single dNTP in a reaction mix results in (relatively inefficient) addition of that nucleotide. This complicates cloning, as the supposedly blunt-ended PCR product often is not, and blunt-endedcloning protocols often do not work or are very inefficient. This can be remedied by incubation of PCR products with T4 DNA pol or Klenow pol, which "polishes" the ends due to a 3'->5' exonuclease activity (Lui and Schwartz, 1992; BioTechniques, 20: 28-30). However, this terminal transferase activity is also the basis of a clever cloning strategy: this uses Taq pol to add a single dT to the 3'-ends of a blunt-cut cloning vector such as pUC or pBluescriptTM, and simple ligation of the PCR product into the now "sticky-ended" plasmid (Marchuk et al., 1990; NAR 19: 1156). Incorporation of Restriction Sites in Primers: Although this may be rendered simple by incorporating the same or different restriction sites at the 5'-ends of PCR primers which allows generation of sticky ends and straightforward cloning into appropriate 18 vectors - these should have AT LEAST two additional bases 5' to the recognition sequence to ensure that the enzymes will in fact recognise the sequence - and it is often found that even when this is done, the efficiency of cutting of fresh product is next to zero. This can sometimes be remedied by incubating fresh product with Proteinase K (to digest off tightly-attached Taq pol), but often is not. A solution to the problem is to use the "Klenow-Kinase-Ligase" (KKL) method: this involves "polishing" products with Klenow, kinasing them to get 5'-phosphorylation (NB: OLIGONUCLEOTIDE PRIMERS NORMALLY HAVE NO 5'-PHOSPHATES!!!), ligating the fragments together to get concatemers, then restricting these with the appropriate restriction enzymes to generate the sticky-ended fragments suitable for cloning (Lorens, 1991; PCR Methods and Applications, 1: 140-141). AND ALWAYS REMEMBER: * WORK CLEAN * TITRATE MAGNESIUM * DON'T USE TOO MUCH TEMPLATE DNA * DON'T USE PCR PRODUCTS IN PCR PREPARATION AREAS * ALWAYS, ALWAYS INCLUDE WATER AND VERY DILUTE POSITIVE CONTROLS IN EVERY EXPERIMENT * WEAR GLOVES * USE PLUGGED TIPS 19 4-Troubleshooting 1. I get (many) longer unspecific products. What can I do? Decrease annealing time Increase annealing temperature Decrease extension time Decrease extension temperature to 62-68º C Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM. Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant. Take less primer Take less DNA template Take less Taq polymerase If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s) Combine some/all of the above 2. I get (many) shorter unspecific products. What can I do? Increase annealing temperature Increase annealing time Increase extension time Increase extension temperature to 74-78º C Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mM Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant Take less primer Take less DNA template Take less Taq polymerase If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s) Combine some/all of the above 3. Reaction was working before, but now I can't get any product. Make sure all PCR ingredients are taken in the reaction (buffer, template, Taq, etc) Change the dNTP solution (very sensitive to cycles of thawing and freezing, especially in multiplex PCR) If you just bought new primers, check for their reliability (bad primer synthesis ?) Increase primer amount Increase template amount Decrease annealing temperature by 6-10º C and check if you get any product. If you don't, check all your PCR ingredients. If you do get products (including unspecific ones) reaction conditions as described above. Combine some/all of the above 20 4. My PCR product is weak. Is there a way to increase the yield? Gradually decrease the annealing temperature to the lowest possible. Increase the amount of PCR primer Increase the amount of DNA template Increase the amount of Taq polymerase Change buffer (KCl) concentration (higher if product is lower than 1000bp or lower if product is higher than 1000bp) Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerol. Check primer sequences for mismatches and/or increase the primer length by 5 nucleotides Combine some/all of the above 5. My two primers have very different melting temperatures (Tm) but I cannot change their locus. What can I do to improve PCR amplification? An easy solution is to increase the length of the primer with low Tm. If you need to keep the size of the product constant, add a few bases at the 3' end. If size is not a concern, add a few bases at either the 3' or the 5' end of that primer. 6. I have a number of primer pairs I would like to use together. Can I run a multiplex PCR with them?. How? Very likely, yes. Try amplify all loci seaprately using the same PCR program. If one of the primer pairs yields unspecific products, keep the cycling conditions constant and change other parameters as mentioned above (#1 and #2). Mix equimolar amounts of primers and run the multiplex reaction either in the same cycling conditions or by decreasing only the annealing temperature by 4º C. If some of the loci are weak or not amplified, read below !! 7. How many loci can I amplify in multiplex PCR at the same time? Difficult to say. The author has routinely amplified from 2 to 14 loci. Literature describes up to 25 loci or so. 8. One or a few loci in my multiplex reaction are very weak or invisible. How can amplify them? The first choice should be increasing the amount of primer for the "weak" loci at the same time with decreasing the amount of primer for all loci that can be amplified. The balance between these amounts is more important than the absolute values used !!. Check primer sequences for primer-primer interactions 9. Short PCR products in my multiplex reaction are weak. How can I improve their yield? 21 Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.52mM Decrease denaturing time Decrease annealing time and temperature Decrease extension time and temperature Increase amount of primers for the "weak" loci while decreasing the amount for the "strong" loci. Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerol Combine some/all of the above 10. Longer PCR products in my multiplex reaction are weak. How can I improve their yield? Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mM Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant. Increase denaturing time Increase annealing time Decrease annealing temperature Increase extension time and temperature Increase amount of primers for the "weak" loci while decreasing the amount for the "strong" loci Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerol Combine some/all of the above 11. All products in my multiplex reaction are weak. How can I improve the yield? Decrease annealing time in small steps (2º C) Decrease extension temperature to 62-68º C Increase extension time Increase template concentration Increase overall primer concentration Adjust Taq polymerase concentration Change KCl (buffer) concentration, but keep MgCl2 concentration at 1.5-2mM Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant. Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerol Combine some/all of the above 12. Unspecific products appear in my multiplex reaction. Can I get rid of them somehow? 22 If long: increase buffer concentration to 1.2-2x, but keep MgCl2 concentration at 1.5-2mM If short: decrease buffer concentration to 0.7-0.9x, but keep MgCl2 concentration at 1.5-2mM Gradually increase the annealing temperature Decrease amount of template Decrease amount of primer Decrease amount of enzyme Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerol If nothing works: run PCR reactions for each (multiplexed) locus individually, using an annealing temperature lower than usual. Compare the unspecific products for each locus tested with the unspecific products seen when running the multiplex PCR. This may indicate which primer pair yields the unspecific products in the multiplex reaction. Combine some/all of the above (Note: primer-primer interactions in multiplex PCR are usually translated into lack of some amplification products rather than the appearance of unspecific products) 5-The applications of PCR PCR is such a straightforward procedure that it is sometimes difficult to understand how it can have become so important in modern research. First we will deal with its limitations. In order to synthesize primers that will anneal at the correct positions, the sequences of the boundary regions of the DNA to be amplified must be known. This means that PCR cannot be used to purify fragments of genes or other parts of a genome that have never been studied before. A second constraint is the length of DNA that can be copied. Regions of up to 5 kb can be amplified without too much difficulty, and longer amplifications - up to 40 kb - are possible using modifications of the standard technique. However, the >100 kb fragments that are needed for genome sequencing projects are unattainable by PCR. What are the strengths of PCR? Primary among these is the ease with which products representing a single segment of the genome can be obtained from a number of different DNA samples. We will encounter one important example of this when we look at how DNA markers are typed in genetic mapping projects. PCR is used in a similar way to screen human DNA samples for mutations associated with genetic diseases such as thalassemia and cystic fibrosis. It also forms the basis of genetic profiling, in which variations in microsatellite length are typed. 23 The use of microsatellite analysis in genetic profiling. In this example, microsatellites located on the short arm of chromosome 6 have been amplified by the polymerase chain reaction. The PCR products are labeled with a blue or green fluorescent marker and run in a polyacrylamide gel, each lane showing the genetic profile of a different individual. No two individuals have the same genetic profile because each person has a different set of microsatellite length variants, the variants giving rise to bands of different sizes after PCR. The red bands are DNA size markers. Image supplied courtesy of PE Biosystems, Warrington, UK, and reproduced with permission. A second important feature of PCR is its ability to work with minuscule amounts of starting DNA. This means that PCR can be used to obtain sequences from the trace amounts of DNA that are present in hairs, bloodstains and other forensic specimens, and from bones and other remains preserved at archaeological sites. In clinical diagnosis, PCR is able to detect the presence of viral DNA well before the virus has reached the levels needed to initiate a disease response. This is particularly important in the early identification of viral-induced cancers because it means that treatment programs can be initiated before the cancer becomes established. These are just a few of the applications of PCR. The technique is now a major component of the molecular biologist's toolkit and we will discover many more examples of its use in the study of genomes. Although PCR was first developed only a decade and a half ago, the simplicity and the versatility of the technique have ensured that it is among the most ubiquitous of molecular genetic methodologies, with a wide range of general applications PCR enables rapid amplification of template DNA for screening of uncharacterized mutations Because of its rapidity and simplicity, PCR is ideally suited to providing numerous DNA templates for mutation screening. Partial DNA sequences, at the genomic or the cDNA level, from a gene associated with disease, or some other interesting phenotype, immediately enable gene-specific PCR reactions to be designed. Amplification of the appropriate gene segment then enables rapid testing for the presence of associated mutations in large numbers of individuals. By contrast, cell-based DNA cloning of the gene from numerous different individuals is far too slow and labor-intensive to be considered as a serious alternative. Typically, the identification of exon-intron boundaries and sequencing of the ends of introns of a gene of interest offers the possibility of genomic mutation screening. Individual exon-specific amplification reactions are developed by designing primers which recognize intronic sequences located close to the exon-intron boundary. The resulting PCR products are then analyzed by rapid mutation-screening methods, in which the optimal size for mutation screening is usually about 200 bp. Conveniently, the average size of a human exon is about 180 bp but, in the case of very large exons, it is usual to design a series of primers to generate overlapping exonic products. PCR can also quickly provide amplified cDNA sequences for mutation screening. Such cDNA mutation screening may be the only way in which mutations can be screened if the exon-intron organization of a gene has not been established. To do this, mRNA is isolated from a convenient source of tissue, such as blood cells, converted into cDNA using reverse transcriptase and the cDNA is used as a template for a PCR reaction. This version of the standard genomic PCR reaction is consequently often referred to as RT-PCR (reverse transcriptase-PCR). Clearly, the 24 method is ideally suited to genes expressed at high levels in easily accessible cells, such as blood cells. However, as a result of low level ectopic transcription of genes in all tissues, it has also been applied to transcript analysis of genes which are not significantly expressed in blood cells, such as the dystrophin (DMD) gene (Chelly et al., 1989). PCR products for gene mutation screening are obtained from genomic DNA using intronspecific primers flanking exons or by RT-PCR. (A) Genomic DNA. Exons 1–4 can be amplified separately from genomic DNA using pairs of intron-specific primers 1F + 1R, 2F + 2R, etc. (B) RT-PCR. This relies on at least some mRNA being present in easily accessible cells such as blood cells, permitting conversion to cDNA. The cDNA can then be used as a template for pairs of exon-specific primers (1F+1R, 2F+2R, etc.) to generate overlapping DNA fragments. PCR permits rapid genotyping for polymorphic markers Restriction site polymorphisms (RSPs) result in alleles possessing or lacking a specific restriction site. Such polymorphisms can be typed using Southern blot hybridization. A DNA probe representing the locus is hybridized against genomic DNA samples that have been digested with the appropriate restriction enzyme and size-fractionated by agarose gel electrophoresis. The resulting RFLPs have two alleles corresponding to the presence or absence of the restriction site. As a convenient alternative to RFLPs, PCR can type RSPs by simply designing primers using sequences which flank the polymorphic restriction site, amplifying from genomic DNA, then cutting the PCR product with the appropriate restriction enzyme and separating the fragments by agarose gel electrophoresis. 25 8- PCR Glossary AFLP AFLP Amplified Fragment Length Polymorphism Genomic DNA is digested with MseI-EcoRI and the MseI-EcoRI fragments are PCR amplified to reveal length polymorphism. Asymmetric PCR Preferential PCR amplification of one strand of DNA by lowering the concentration of one primer. Bisulfate sequencing Used to detect CpG methylation in genomic DNA. Sodium bisulfate converts unmethylated cytosine to Uracil. Comparison of sodium bisulfate treated and untreated DNA reveal methylated cytosines. DD-PCR Differential Display PCR A technique of comparing gene expression between related cells. Arbitrary primers are used to amplify 3'-ends of subpopulations of polyA mRNAs. DOR PCR Degenerate Oligonucleotide-primed PCR (DOR PCR) Degenerate PCR Degenerate PCR primers used to amplify unknown DNA sequences based on similar known sequences. Forensic PCR VNTR locus are PCR amplified to compare DNA samples from different sources. Hairpin PCR A method of error-free DNA amplification for mutation detection. It first converts a DNA sequence to a hairpin. True mutations will maintain the hairpin structure during amplification while PCR errors will disrupt the hairpin structure. In situ PCR PCR on fixed cells. DNA or RNA is immobilized in their subcellular locations. Long PCR Amplification of long target DNA sequences. MSP PCR Methylation Specific PCR Used to detect CpG methylation in genomic DNA. Sodium bisulfate converts unmethylated cytosine to Uracil. Comparison of sodium bisulfate treated and untreated DNA reveal methylated cytosines. PCR-RFLP See Restriction Fragment Length Polymorphism (PCR-RFLP) 26 By designing primers that will introduce or destroy a restriction site for one of the alleles, the PCR products for SNP alleles can be distinguished by restriction fragment lengths. Competitive PCR (cPCR) A method for quantify DNA using real-time PCR. A competitor internal standard is co-amplified with the target DNA. Target is quantified from the melting curves of the target and the competitor. PCR-Single Strand Conformational Polymorphism (PCR-SSCP) PCR followed by SSCP detection of point mutations. Single Strand Conformational Polymorphism (SSCP) Electrophoresis separation of single-stranded nucleic acids based on differences in sequence. Single base mutations may be detected because single base mutations may disrupt secondary structure of the SS DNA and leads to changes in mobility through the gel. Rapid Amplification of cDNA Ends (RACE) Used for cloning full-length 5' or 3' ends of a cDNA. An adapter sequence is added to either 5' or 3' ends of cDNA. The two PCR primers are either specific to the adapter or specific to known sequences of cDNA. RAPD Random Amplified Polymorphic DNA (RAPD) Random primers are used to amplify genomic DNA. Patterns of bands may be different for individuals in a population or closely related species. Serial Analysis of Gene Expression (SAGE) A technique for profiling gene expression in cells. SAGE characterizes a short segment of DNA, called a SAGE tag, in each expressed gene. cDNA is digested with the restriction enzyme Nla III, ligated to a linker, then digested with BsmF1, an enzyme that cleaves 10-14 bases from the Nla III cut. SAGE tags are then concatenated, cloned, and sequenced. Touchdown Decreasing the annealing temperature by 1 degree every one or second cycle. 27