Agarose, PAGE and restriction digestion
Determination of PCR products by agarose gel electrophoresis
One of the simplest methods for the estimation of the efficiency of amplification of PCR fragments is agarose gel electrophoresis. Different concentrations of agarose can be used, depending on the expected size of the product. The following table shows different concentrations of agarose in relation to product size as described by Sambrook et al (1989).
Table 1 Range of separation in gels containing different amounts of agarose
Using a calibrated pipette, 5ml of each of the PCR products were added into each well of a microtitre plate followed by the addition of 1ml of 6x loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 15% Ficoll in water). Agarose gel (1%) was prepared by dissolving 1g agarose in 100mls 1x TAE buffer (40mM Tris; 20mM acetate; 2mM EDTA; pH 8.1) by heating. After cooling to around 60ºC, 5µl ethidium bromide were added to the gel followed by pouring into a casting tray, creating wells with a comb, and left to solidify for 45 minutes. The freshly prepared gel was then placed in the electrophoresis tank and each sample was loaded into a separate well in the gel. Seven microlitres of the size marker fX 174 DNA digested with HaeIII, were loaded into the first well of each row. This marker is used for comparison with the PCR product to ensure that the fragment obtained is of the expected size.
Electrophoresis was carried out in 1x TAE (40mM Tris; 20mM acetate; 2mM EDTA; pH 8.1) at a voltage of 150V and variable current for 15 minutes. The gel was scanned using UV irradiation and photographed (Polaroid DS-34).
Restriction Enzyme Digestion
One of the easiest ways to detect a polymorphism in molecular biology is by using restriction enzyme digestion of the PCR product. If a polymorphism either introduces or abolishes a cut site for a specific endonuclease. Table 2-5 shows the specific endonucleases used to detect the polymorphisms studied.
The PCR products (10ml) were digested using 4.5 U of appropriate restriction enzymes according to manufacturer’s instructions (New England Biolabs, Beverly, MA, USA). A reaction mixture was prepared for each tube by first pipetting 2.5ml of the appropriate 10x NE buffer (supplied with each enzyme), using calibrated pipettes. This volume is equivalent to one tenth of the final volume of the reaction mixture. Table 2 shows the components for each NE buffer used. Four units of the appropriate enzyme were added into each tube that was made up to 15ml by the addition of sterile distilled water. 10 µl from each PCR product were added into corresponding tubes to make up a total reaction volume of 25ml, after which mineral oil was added to prevent evaporation. Positive and negative controls were included with every batch of samples done. A known homozygote for the cleaved site was used as positive control while a sample with no enzyme added to the reaction mixture containing the PCR product was used as a negative control. All tubes were then incubated overnight at the appropriate temperature as recommended by supplier (New England Biolabs, Beverly, MA, USA). After incubation all tubes were stored at -20°C until analysed by agarose or polyacrylamide gel electrophoresis (PAGE).
Table 2 Components of NE Buffers (1 X) used for RFLP
Detection of the digested products by electrophoresis
Agarose Gel Electrophoresis
To detect the genotypes, the digested PCR products were analysed using agarose gel electrophoresis. As already described, the percentage concentration of the gel used for electrophoresis was determined according to the size of the expected fragments, and ranged from 1.5% to 2.0%.
Agarose gel was prepared as previously described (Section 2.4.2). The digested products were allowed to thaw, after which 15ml were mixed with 3ml of 6x loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 15% Ficoll in water) and loaded on the gel. In the first well of each row, 7ml of fX174 DNA digested with HaeIII were loaded as a size marker. Electrophoresis was carried out in 1x TAE buffer (40mM Tris; 20mM acetate; 2mM EDTA; pH 8.1) at a voltage of 150V and a variable current for 60 minutes. After electrophoresis was complete, the gel was illuminated using UV irradiation and photographed (Figure 1).
Figure 1. Agarose gel electrophoresis (2%) of VDR FokI digest. Lane A: Molecular weight marker; B: CT heterozygote; C: TT homozygote; D: CC homozygote
Polyacrylamide Gel Electrophoresis (PAGE)
Polyacrylamide gel electrophoresis was used to detect fragments of less than 100bp with differences of only 20 bp between each fragment, as it has a much higher resolution than agarose gel. Twenty millilitres of a 10% non-denaturing polyacrylamide gel was freshly prepared by first adding 5ml of 40% acrylamide/bisacrylamide (38:2 w/w) solution into a 100 ml glass beaker. Two millitres of 10x TAE were added followed by 140µl of freshly prepared 10% (w/v) ammonium persulphate, and made up to 20 ml by deionised water.
Gel glass plates were prepared by placing 1mm spacers at the edges of a large glass plate and the notched plate was placed onto it. The glass plates were then sealed by using a gel casting unit. Just prior to pouring the acrylamide solution, 30µl of TEMED were added. This acts as a catalyst for the polymerisation reaction. The gel was then poured slowly and side ways down the inside of the glass plates taking care not to introduce air bubbles. The comb was inserted and the gel was allowed to polymerize for 45 minutes under UV irradiation. Following polymerization the comb was removed carefully and the gel placed in a vertical electrophoresis tank filled with 1x TAE buffer.
Prior to sample loading, 10µl of each sample were added to 2µl of 6x loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 15% Ficoll in water) in a microtitre plate and mixed well. Using a gel loading pipette 12µl of sample mix were loaded carefully to the bottom of each well. The lid was replaced and electrophoresis was carried out for 120 minutes at a voltage of 100V with variable current.
When electrophoresis was complete the gel was removed from the tank, unclamped and the plates separated carefully as not to damage the gel. The gel was then stained with ethidium bromide, rinsed with water and visualised under UV irradiation and photographed (Figure 2).
Figure 2. 9% PAGE of MTHFR C677T SNP. Lane A: Marker; B: negative cnt; C: TT homozygote; D and E: CC homozygotes; F: CT heterozygote (genotyping according to Jorgensen et al., 2002)