PCR AMPLIFICATION

DNA amplification via Polymerase Chain Reaction (PCR) has become an increasingly important tool o f molecular biology. A number o f different PCRs were performed for the research in this thesis, either to amplify a DNA fragment, a polymorphic region [such as fragments containing poly ‘CA ’ microsatellites or trinucleotide repeats (TNR)] or from bacterial colonies (colony PCR). The region o f interest was amplified from either genomic DNA or from cloned and enriched DNA. To perform these, either a standard protocol, applicable to most PCRs or a specific protocol, applicable to a specific pair o f

Methods and Materials primers was used. PCR amplification o f polymorphic regions was mostly performed radioactively, unless otherwise stated. When PCR amplification failed to generate products or produced spurious bands, the conditions were optimised by varying the reaction conditions. Likewise, the cycling conditions were also varied. A ‘Taguchi’ method was used to optimise PCR conditions. All the necessary information are provided in the sections below. Refer to Innis et al. (1990) for more detailed accounts o f PCR amplification protocols and applications.

2.4.1 Design of primers

Primers used to amplify specific target regions were designed in the range o f 17-25 nucleotides in length. Non-repetitious template DNA sequence was used to design the primers, which reduces mispriming, unless in some circumstances where it was unavoidable. Primers were designed such that the percentage GC content was within the range o f 50 to 60%, but in the case o f G+C rich template DNA sequence, the G+C content o f the primers was higher. Primers were also designed preferably with a ‘C ’ or ‘G ’ base at the 3’-end o f the primer, as this provides a stronger attachment to the template. The melting temperature (Tm) o f the primers was calculated as follows: 2°C was assigned for a A or T nucleotide base and 4°C for a G or C nucleotide base, and added together (Thein and Wallace, 1986).

2.4.2 The standard PCR amplification protocol

The standard condition will amplify most target sequences. These conditions were used initially to amplify a target region specified by a primer pair; if the reaction failed to generate products o f interest then other PCR amplification protocols were used. The standard composition o f a PCR reaction consisted o f Ix NH4 [750mM Tris-HCl/ 200mM (NH4)2S0 4, pH9.0 at 25°C, Bioline] or KCl reaction buffer (lOOmM Tris-HCl/ 500mM KCl, pHS.S at 25°C, this buffer has M gCl2 added to it, at a Ix final concentration o f buffer, the final concentration o f MgCl2 will be 1.5mM, Bioline),

0.5)liM o f each primer, 0.2mM dNTP (dATP, dCTP, dGTP and dTTP), 1.5mM M gCb (MgCl2 was not added if the KCl buffer was used), 50-100ng o f genomic DNA or template DNA and 0.1-0.3 unit o f Bioline BioTb^ DNA polymerase. The final volume o f the reaction was made up with sterile distilled water (SDW). The total volume o f the

Methods and Materials reaction varied depending on the volume o f the PCR product required.

The standard PCR cycling condition consisted o f an initial denaturing step at 95°C for 5 minutes followed by 35 cycles o f denaturing at 95°C for 30 seconds, annealing at Ta (annealing temperature specific for each primer pair, the Ta used was usually 2°C lower than the lowest figure o f Tm for the primer pair) for 30 seconds, elongation at 72°C for 30 seconds and a final extension step at 72°C for 2-10 minutes. The elongation time was increased according to the size o f the fi-agment being synthesised. Typically for fragments less than 1Kb elongation was performed for 30 seconds. For larger fragments, elongation was performed for a maximum o f 1 minute. PCR cycling was performed either on an Omni thermal-cycler blocks or on a Perkin-Elmer Cetus thermal cycler model 9600 or 2400. 0.5ml Eppendorf tubes were used for the Omni thermal-cycler and the PCR reaction mixture was always covered with mineral oil to prevent evaporation during the cycling process. 0.2ml thin wall tubes or strips o f 8 tubes (CAMLAB) were used for the Perkin Elmer thermal-cycler. Mineral oil was not added to the PCR reaction mixture as these machines have heated lids, which prevent evaporation.

2.4.3 PCR conditions for high G + C rich templates

Application o f the standard PCR protocol was unsuccessful for regions such as the dopamine D4 receptor gene, a gene with a very high G + C content {<15% ). G + C rich sequences are able to form stable secondary structures thereby inhibiting the progression o f DNA polymerases along the template during the amplification process. This leads to the generation o f small products caused by incomplete amplification. Furthermore primers designed for G + C rich templates are themselves high in G + C content and therefore bind non-specifically to the DNA template. The secondary structures also prevent the binding o f primers. This non-specific binding generates spurious PCR amplification products.

To reduce the formation o f secondary structures and non-specific binding o f the primers, the PCR amplification was performed with high dénaturation and annealing temperatures. Under such circumstances heat stable thermal DNA polymerases, such as native Pju (Stratagene), Tth (Pharmacia Biotech) and BioTaq (Bioline), or a combination o f these, were used with the supplied reaction buffers. Perfect Match^*^ polymerase enhancer

Methods and Materials (Stratagene), an additive was used to increase the specificity o f some difficult reactions. It functions by destabilising many mismatched primer template complexes that would otherwise result in a heterogeneous population o f amplified molecules

Cosolvents or dénaturants such as DMSO, DTT, formamide, and glycerol were also used in the PCR reaction to reduce the formation o f secondary structures in the template. These solvents also facilitate the dénaturation o f the template and primer/ template duplexes, thus enhancing the specificity o f the reaction. DMSO and glycerol function b y effectively lowering the melting and strand separation temperatures (Wong et a l, 1991; Chester and Marshak, 1993). The reaction composition for each primer pair was also optimised by varying the primer concentration and the final concentration o f MgCl2 in the reaction between 1.5mM and 4mM. Therefore the PCR composition for each primer pair varied and the conditions are discussed in the relevant sections.

The ‘touchdown’ PCR cycling protocol (Don et al., 1991) was applied to these PCRs. This procedure incorporates high annealing temperatures in the initial cycling reactions which reduces incorrectly annealed primers and mis-extension o f incorrect nucleotides at the 3’ end o f primers. This procedure generates a small amount o f amplified PCR product o f interest in the first cycle. This will then be used in preference to the genomic DNA present in the reaction, as a template for the next cycle o f amplification which would have a lower annealing temperature. After a small number o f cycles (5-10) enough template would be generated to carry out the normal PCR cycling procedure. The template amplified by the touchdown cycle would be preferred over the more complex genomic DNA by the unbound primers.

This protocol consists o f an initial dénaturation step at 98° for 5 minutes. This is followed by a number o f cycles, o f which the first cycle consists o f dénaturation at 98°C for 30 seconds, an annealing step, with a high Ta, ideally 10 degrees above the specific Ta, for 30 seconds and elongation at 72° for 30 seconds. The next cycle will be the same, except the Ta would be reduced either by 1 or 2 degrees. The next few cycles were performed in this touchdown manner until the specific Ta for the primer pair is reached. For example, for a primer pair with Ta o f 70°C, 10 cycles were performed. The first cycle was performed at Ta o f 80°C, followed by the next at Ta o f 79°C and so on until the last cycle which is performed at 71°C. Finally the following cycling protocol

Methods and Materials consisting o f 35 cycles o f 98°C for 30 seconds, Ta for 30 seconds and 72°C for 30 seconds was followed. The PCR cycling was performed on the Perkin Elmer Cetus thermal cycler 9600 or 2400.

2.4.4 The modified ‘Taguchi’ method

The modified ‘Taguchi’ method (Cobb and Clarkson, 1994) was used to optimise PCR conditions for difficult primers. The normal optimisation process for a primer pair relies on the sequential investigation o f each reaction variable, an approach that leads to a large number o f optimisation experiments in order to include all possible combinations. This modified ‘Taguchi’ strategy circumvents many o f the problems associated w ith conventional optimisation strategies. A brief description o f the method is given below, refer to Cobb and Clarkson (1994) for a more detailed explanation.

In this method, factors which are thought likely to effect the PCR process are arranged into an orthogonal array. Three different final concentration o f primer (for example 7.5pmol, ISpmol and 30pmol, lp.M primer concentration = lpmol/p.1), DNA template (for example lOOng, 200ng and 600ng), MgCl] (for example 0.5mM, Im M and 5mM ) and dNTP (O.OSmM, 0.2mM and 0.4mM) are chosen such that they are sufficiently separated so that their effects on the reaction can be determined. Nine reactions are set up with various combinations o f these four variables, such that all possible combinations o f the four variables are used in each o f the nine reaction. Thereby optimising the PCR over nine reactions as opposed to 81 separate reactions when using conventional methods.

2.4.5 a-^^P-dCTP incorporation PCR

PCR amplification o f regions containing polymorphic dinucleotide and TNRs was performed radioactively by incorporation o f a-^^P-dCTP and analysed on denaturing polyacrylamide gels. The PCR reaction is set up as for a standard reaction (see section 2.4.2) but with two variations. One o f which is the addition o f 0.03p,l o f a-^^P-dCTP (lOmCi/ml, 6000Ci/mmol) per lOpl o f reaction volume. The second variation is the use o f dNTPs with dCTP at a 1/10 o f the concentration o f dATP, dGTP and dTTP. Hence 2mM dNTPs stocks were made with 2mM dATP, dGTP, dTTP and 0.2mM dCTP.

Methods and Materials Standard cycling protocol was used (see section 2.4.2).