H eteroduplex analysis

The PCR products o f normal and mutant alleles can be separated from each other using non ­ denaturing gel electrophoresis. Resolution is based upon conformational differences that occur in the DNA molecule as a result o f the mutant allele which can be a result o f insertions, deletions or base pair mismatches. Heteroduplex analysis involves the detection o f conformational differences in double stranded DNA molecules as opposed to the detection o f single stranded conformational polymorphisms (SSCP). Heteroduplex DNA was generated by standard PCR amplification. Amplification occurs between homologous DNA segments as well as across the segment containing the mutant allele. By denaturing these sample and allowing to

cool to room temperature, double stranded DNA is formed between the identical

complementary strands (homoduplexes) and also between strands o f the 2 different amplified

segments (heteroduplexes). These heteroduplexes migrate at a slower rate on acrylamide than the corresponding homoduplexes.

MDE gels were routinely used for the detection of hetroduplexes. The size range o f PCR products analysed in this study ranged from 100 - 3 0 0 bp in size (within the optimal size range for resolution). The electrophoresis apparatus (using 40 cm X 2 0 cm glass plates) was vertically assembled according to manufactures instructions (J.T. Baker, USA) and clamped within the casting tray. A 100 ml gel solution was prepared by the addition o f 50 ml MDE gel solution, 6 ml 1 OX TBE, 44 ml sterile distilled water, 4 0 0 |il 10 % Ammonium per sulphate

(APS) and 4 0 |il TEMED. Prior to addition of APS and TEMED, 2 ml was removed, to which 30 |xl APS and 12 |ll1 TEMED added, and gently poured between the plates to form a seal at the base. On setting, the remaining gel was, mixed and poured between the plates avoiding the formation o f air bubbles. On insertion o f an appropriate comb, the gel was allowed to polymerise for 1 hour following which the comb was removed and 0.6X TBE added to the upper and lower buffer reservoirs. On rinsing of the wells, 10 p.1 samples containing 100 - 2 0 0 ng PCR product in 2 pi loading buffer containing xylene cyanol and bromophenol blue was loaded in to each well. Electrophoresis was allowed to occur for - 1 6 hours at 180 volts with the xylene cyanol and bromophenol blue being indicators of resolution. The gels were stained with ethidium bromide ( 0.5 pg/ ml) and UV photographed.

2.4 A standard PC R protocol

These are the parameters and reaction conditions used throughout the course of this work and relevant alterations to this protocol are stated where necessary.

PCR reactions were typically carried out in 25 |il volumes consisting of 1 X PCR buffer (lOX N H4 buffer, Bioline), 1.5 mM MgCl2 (Bioline), 200 |iM each dNTP (Promega), 7.5 pmoles

each primer, 0.3 units of Taq DNA polymerase (Bioline) and -100 ng template DNA. The temperature cycling profile consisted of an initial dénaturation at 94 °C for 4 minutes followed by 30 cycles of dénaturation at 94 °C for 30 seconds, annealing for 30 seconds (annealing temperature being primer dependent) and extension at 72 °C for 30 seconds. The reaction was completed with a final cycle of extension at 72°C for 2 minutes. A Hybaid Omnigene thermal cycler or a Perkin-Elmer Cetus system 2400 or 9600 thermal cycler were consistently used for all PCR analyses.

2.4.1 U se o f ‘hot starts’, ‘touch down’ PCR, the T aguchi m ethod and ‘Perfect M atch ’

It was sometimes necessary to modify the PCR protocol to improve the quality of the product. This initially involved experimenting with various temperatures above and below the calculated annealing temperatures of primer pairs or the alteration in MgCl] concentration. Most often a ‘hot start’ was adequate to remove nonspecificity, this was essentially the same protocol as above but with the addition of Taq polymerase after the initial cycle o f dénaturation. ‘Touchdown’ PCR was based on the initial amplification o f a perfectly matched target at a high stringency followed by the product of this amplification being favoured as the template for subsequent rounds of amplification. The cycling parameters were set so that initial cycling occurred at a temperature setting above the annealing temperature o f the primer pair and with each subsequent cycle the temperature was reduced by 1 °C until it reached a value slightly below the calculated annealing temperature of the primers. It was at this lower temperature that the remaining cycles were carried out. The Taguchi m ethod was primarily designed to optimise the PCR conditions for a given primer pair. It is based on 9 PCR reactions each containing varying amounts of DNA template, primer concentrations, dNTPs and MgCI] . The products were visualised by agarose gel electrophoresis and scored from 0-10 based on the quality of the product for each of the four variables. The score obtained was applied to an equation from which the highest value obtained for each variable was indicative o f the most suitable concentration for that particular primer pair (Cobb and Clarkson, 1994). The ‘perfect match k it’ supplied by Invitrogen, UK is also aimed at optimising PCR conditions for primer pairs and is based on the use of specific buffers containing varying concentrations of PCR variables. Reactions were performed on instructions provided by the supplier.

Methods and Materials