ESTADÍSTICAS SOBRE EL CONOCIMIENTO DE LA CONTAMINACIÓN

Capillary gel electrophoresis (CGE) is a technique that has recently begun to be used for

the analysis o f PCR products (M cCord el al, 1993b; Srinivasan el al, 1993; Lu et al,

1994). Many o f these applications are based on the fact that CGE separations o f nucleic

acids can have very fine resolution (4 bp resolution has been reported (M cCord et al,

Chapter 3. Development o f QPCR Assays

system such as laser induced fluorescence (LIF) to detect very low amounts of DNA (McCord et al, 1993b; Srinivasan et al, 1993). The advantage o f CGE-LIF with respect to PCR quantitation is that its sensitivity is such that PCR products can be measured whilst the PCR is still within the linear portion of the reaction. With adequate controls this allows direct quantitation of the starting number of copies of template in a PCR.

In these experiments CGE was investigated as an alternative to standard agarose gel electrophoresis for quantitation of PCR products. The samples used for analysis by CGE were the same as those used in construction of the calibration curves (Sections 3.2.3 and 3.2.5), the principle of quantitation being the same, that is, based on the use of a competitive internal standard. However, it was hoped that CGE, once a method was established, would offer certain advantages over agarose gel electrophoresis (see Figure 3.20): i) analysis by CGE allows direct quantitation of DNA fragments in solution without the additional densitometry step; ii) CGE analysis can be readily automated since there is no need to pour fresh slab gels for each series of samples and because the instrument used (see Section 2.3.3) has an auto sampler allowing up to 40 samples to be analysed at a time; and iii) CGE analysis uses UV detection of DNA fragments, hence there is no requirement for ethidium bromide.

Agarose Gel Electrophoresis

PCR'

Run agarose gel

1

Take image of gel under UV Download image to disk

I

Analyse stored image using Gelbase software

I

Relative band intensities

Load CE, run

Capillary Gel Electrophoresis

Relative peak intensities

Calculate quantity of target DNA

FIGURE 3.20 Comparison of agarose gel electrophoresis and CGE for quantitation of PCR products

The results of CGE analysis of PCR product samples were promising but ultimately frustrating. Although it was found that all of the IS and pQR701/126 fragment bands could be adequately resolved from each other and from the components of the PCR mixture (which do show up on a chromatogram due to their UV absorbance), it was not

possible to achieve enough data to be truly convincing because of equipment failings. The main problem was that the capillary used (at a cost of £400) was liable to breakage, which occurred as the capillary entered the instrument. Other problems such as an increase in the baseline noise as the running buffer ages and as the number of samples analysed increased were overcome by the use of fresh buffer and the introduction of a 5 minute pressure rinse step between runs. Some separations achieved by CGE are shown in the Figures 3.21 and 3.22.

Analysis of the peak area ratios (IS/pQR) obtained by agarose gel electrophoresis and CGE indicate that the two techniques gave similar results (within 10%). However, it was not possible to fulfil the aim of constructing a calibration curve using CGE, due to the problems mentioned earlier. The sensitivity of the two techniques were also found to be approximately equivalent, although with CGE the sample volume loaded is much less (<lpL) than that used in agarose gel electrophoresis (7.5pL). The smaller sample volume required in CGE allows more re-runs in the event of an inconclusive result or alternatively introduces the possibility of running smaller volume (and cheaper) PCRs (see Section 7.1.4).

Comparison of the two techniques in terms of the time taken to give a final result is not straight forward. This is because CGE runs samples consecutively whereas up to 40 samples can be run in parallel on an agarose gel. Therefore although it takes only 20 minutes to run one sample by CGE and there is no time required for subsequent analysis, whilst a gel takes 2 hours plus analysis time, CGE might not always be faster.

TABLE 3.12 Comparison of time taken from sample loading to results availability using CGE and

No. of samples Turnaround time - CGE (min) Turnaround time - AGE (min)

1 2 0 135

5 1 0 0 145

1 0 2 0 0 170

Chapter 3. Development o f QPCR 0.010 OG 0.008 0.006 CO O X 0.004 0.002 0.000 -0.002 20 10 15

run time (min)

FIGURE 3.21 CG E Separation o f Phi-X 174 RF DNA Hac 111 D igest (Test Mix). Peaks identified; OG; orange G (m arker dye): 1; 72 bp: 2; 118: 3: 194: 4; 234: 5; 271: 6; 281: 7; 301: 8; 603: 9; 872: 10, 1078: 11; 1.353. 0.004 0.003 IS(A) DNA Ç 0.002 &

^

run time (min)

FIGURE 3.22 CG E C hrom atogram o f PCR of pQ R126 and 1S(A). U npurified PCR products loaded onto CGE. PCR com ponents that absorb at 254 nm include proteins, prim ers and prim er/dim er products.

However, since the CGE has an autosampler, it is possible to leave samples running overnight. In addition the 'hands-on' time for CGE is virtually nil irrespective o f the number o f samples, whereas for agarose gel electrophoresis the hands-on time for 10 samples is estimated at 50 minutes and for 40 samples is 200 minutes.

In conclusion it can be said that CGE is potentially a very useful analysis technique for this type o f quantitation. However, it has not been possible to validate the quantitation to any great extent, so all subsequent experiments were carried out using agarose gel electrophoresis and densitometry.