To provide sufficient DNA for mutation analysis and sequencing it was necessary to amplify samples using the polymerase chain reaction as described below.
2.2.2(a). Oligonucleotides.
Oligonucleotides used as primers for PCR are detailed in full in 2A.9. along with respective optimal annealing temperatures. Approximate melting temperatures were initially estimated using the following formula:
Tm = 69.3 + (GC x 0.41) - (650 / L)
Where ‘GC’ is the percentage nucleotides that are either guanine or cytosine, and L is the number of nucleotides in the primer.
Optimal annealing temperatures were determined by experimentation with temperatures either side o f this original estimate. All oligonucleotides were supplied
by Oswel DNA services as working solutions o f approximately 50pM/pl concentration.
2.2.2(b). Amplification reactions.
Polymerase chain reactions were set up on ice in a laminar flow cabinet using dedicated Gilsen pipettes and sterile pipette tips. The final concentration or volume of reagents were as follows 25pM of each primer, 0.2mM o f each 2 ’-deoxynucleoside 5’-triphosphate (dATP, dGTP, dCTP, dTTP), 5pl of 10 X enzyme reaction buffer (HT Biotechnologies), and 0.5 units of Taq DNA polymerase (HT Biotechnologies), in a total volume o f SOpl. Reaction mixtures were added to 250ng-500pg of genomic DNA in a microcentrifuge tube and were overlayed with 50pl of mineral oil (Sigma). Thermal cycling was carried out using an Omnigene PCR machine (Hybaid).
Amplification of DNA fragments from APC, tiMSH2, and microsatellite loci for SSCP analysis was achieved using the following set o f conditions:
1- 4.50 minutes at 94°C (dénaturation)
2- 0.50 minutes at 94°C (dénaturation)
3- 0.75 minutes at the appropriate temperature* (annealing)
4- 0.75 minutes at 72°C (elongation)
5- 10.0 minutes at 72°C (elongation)
*See section 2A.9.
Steps one and five only took place once while steps 2-4 were repeated 35 times. Amplification of large APC fragments for PTT involved essentially the same programme the only difference being an increase in the duration o f step four to 1.5 minutes.
2.2.2(c). PCR from single cells.
The single cell PCR protocol was similar to standard PCR, however precautions against contamination were considerably more rigorous. These included setting up the PCR in a laminar downflow cabinet in a room with restricted access, the wearing of a clean gown and gloves, filtration of all reagents using 0.22p.m filters (Millipore), and the use o f filter pipette tips to guard against aerosols. The incidence o f contamination was assessed regularly using numerous control blanks (>50), containing PCR reaction mixture but no DNA. Any reagents shown to cause contamination were immediately discarded, even if amplification was only detected in a small proportion o f blanks.
2.2.2(d). Primer extension preamplification (PEP).
As well as amplification o f specific loci from single cells whole genome amplification (WGA) was also employed. This generated sufficient DNA from a single cell to allow multiple subsequent PCRs. Thus it was possible to amplify two APC fragments, one containing a mutation and one containing a polymorphism without performing multiplex-PCR. The WGA protocol used was primer extension preamplification (PEP). 60pl reaction mixtures contained a lysed cell in 5pi o f single cell lysis buffer, 5pi o f neutralising buffer (2A.8(d).), 6pl o f potassium free PCR buffer (2A.8(b).), 15 base oligonucleotide primers of random sequence at a concentration of 33.3mM, 0.1 mM o f each deoxynucleotide (dATP, dGTP, dCTP, dTTP), and 5 units of Taq polymerase. Thermal cycling involved 50 cycles o f the following: a one minute dénaturation step at 92°C; two minutes annealing at 37°C; a temperature ramp of one degree every 10 seconds to 55°C; and finally the temperature was maintained at 55°C for four minutes. PEP products were stored at 4°C for up to one month.
2.2.2(e). Nesting.
Amplification o f the APC gene in single cells followed a nested PCR strategy to improve the specificity o f the reaction and to reduce the risk of contamination
caused by previously amplified DNA fragments. For the outer amplification 45 pi of reaction mixture was added to either single cells suspended in 5pi o f single cell lysis buffer or to 5pl o f PEP product. The reaction mixture for single cells consisted o f 5pl o f neutralising buffer, 5pl of 10 X potassium free buffer, ImM o f each deoxynucleoside triphosphate (dATP, dGTP, dCTP, dTTP), 4pM o f both forward and reverse primers (2A.9(d).), and 1 unit of Taq polymerase, in a final volume o f 45pl.
For amplification o f PEP products neutralising buffer and potassium free PCR buffer were omitted in favour of 5pi of SC-PCR buffer (2A.8(C).). Mixtures were overlaid with mineral oil and amplification performed according to the following cycling parameters:
Codon 1678 polymorphism detection Codon 764 mutation detection
Outer amplification 96°C 0.75 min 61°C 0.75 min X 2 8 72°C 1.50 min Outer amplification 96°C 0.75 min 55°C 0.75 min X 2 8 72°C 1.50 min
After completion o f the outer amplification 2pl o f PCR product was transferred to a fresh microcentrifuge tube and subjected to further amplification. This involved the addition of 48pl o f reaction mixture containing 1 X SC-PCR buffer, ImM o f each deoxynucleoside, 4pM forward and reverse primers situated internal to those used for the first reaction, 1.5mM MgCl2, and 5 units o f Taq polymerase. The
APC codon 1678 polymorphism detection APC codon 764 mutation detection
Outer amplification Outer amplification
96°C 0.75 min 96°C 0.75 min
56°C 0.75 min X 32 65°C 0.75 min X 32
72°C 1.50 min 72°C 1.50 min