John M. S. Bartlett.pdf - Bio-Nica.info

Detection of MIS 305
1. Archival DNA template (1 µL).
2. 1× reaction buffer (1 µL of 10× reaction buffer); because the reaction buffer contained
15 mM MgCl 2 , this gave a reaction concentration of 1.5 mM MgCl 2 .
3. 2 µM reverse and forward primer (1 µL) to give a reaction concentration of 0.2 µM for
each primer.
4. 0.6 1 µL of 25 mM MgCl 2 to give a final reaction concentration of 3 mM MgCl 2 .
5. 0.2 1 µL of 10 mM dNTP to give an reaction concentration of 200 µL each of dATP,
dCTP, dGTP, and dTTP.
6. 0.1 1 µL of 5 units/1 µL hot star Taq polymerase to give 0.5 units per reaction.
7. Volume is made up to 10 1 µL using PCR-grade water.
The reaction was started after a 15-min denaturation of DNA at 95°C. DNA
amplification was performed in a thermal cycler as follows: 45 cycles of denaturation
at 94°C for 30 s; annealing at 47, 50, or 53°C (Table 1) for 30 s and extension at 72°C
for 1 min; followed by a final extension for 15 min at 72°C.
The PCR is repeated using the same DNA template for each of the primer sets. On
removal from the thermal cycler 2 µL of each of the 9 PCR products from each primer
set are combined for each template and mixed thoroughly by vortexing.
3.4. PCR Analysis
1. PCR products are then prepared for gel electrophoresis.
2. The following are combined in a 200-µL DNA/RNA free microfuge tube and vortexed:
.5 µL of combined PCR products; 1 µL of formamide; 0.5 µL of loading buffer (50 mg/mL
blue dextran, 25 mM EDTA); and 0.5 µL of commercial standard (Genescan 400 HD (rox),
ABI Perkin–Elmer, Norwalk, CT).
3. The mixture is then incubated at 96°C for 4 min and cooled on ice.
4. The mixture (1.5 µL) is applied to a 4% arcrylamide/bisacrylamide gel and electrophoresed
for 2 h on an automated laser-activated fluorescent DNA sequencer (Perkin–Elmer ABI
377 sequencer).
5. Fluorescent gel data were collected automatically during electrophoresis and analyzed
using Genescan software. An example of the gel image is shown in Fig. 1. The data gained
from Genescan is then exported into Genotyper and further analyzed.
3.5. Assessment of Allele Loss and Microsatellite Instability
Allele loss should be assessed as described by Dietmaier et al. (10). In heterozygous
individuals, two alleles, that is, two PCR products of different size can be detected in
normal DNA. Because PCR fragments of different sizes are amplified with different
efficiencies, the ratio of allele peak heights is calculated in matched normal and tumor
DNA. Peak heights of the longer length allele peaks are divided by the peak heights of
the shorter length allele peaks (see Note 2). The ratio obtained in tumor DNA divided
by the allele peak ratio of paired normal DNA gives a result range of 0.00 to 1.00 (see
Note 3), that is, (tumor allele 1 peak height/tumor allele 2 peak height)/(normal allele
1 peak height/normal allele 2 peak height).
Theoretically a complete allele loss results is a value of 0 and both alleles retained
results in a ratio of 1. In cases where the shorter length allele is lost the ratio obtained
is greater than 1, this is therefore inverted (1/×) to obtain values within the 0.00 to
1.00 range. A ratio below 0.65 represents an allele signal reduction of 35%, this is
considered to be indicative of allele loss (see Note 4). This limit was chosen because