1m
Applications in Genetics
Influence of the
Amount of PCR
Product on the
Result of the Melting
Point Analysis
Influence of
Different Primers
and Probes on the
Result of the Melting
Point Analysis
Assay Validation on
Different Samples
20.0 -
16.0 -
2
~
....--..
"0
-Il.O -
N
C
"0
8.0 -
~
U
C
~
•. 0 -
U
..,
~
...
Q
::I
0.0 -
~
68
10
Temperature ( C)
Fig. 2. Melting point analysis of artificial complements using hybridization probe set 1. The numbered curves show results obtained with: 1 (complement with one mismatch corresponding to the
methionine genotype) and 2 (complement without mismatch corresponding to the valine genotype). Melting peaks around 58°C and 65°C; -d(F2)/dT, negative derivative of fluorescence with
respect to temperature
sample {Fig. 4b}. However, for the valine homozygous sample, only the asymmetric PCR conditions produced the correct result with a single melting peak of
around 67°C {Fig. Sb, reactions 2-4}.
After PCR amplification of a heterozygous sample, the PCR product was removed
from the capillary and a dilution series of the product was performed using a PCR
master mix without DNA as diluent. The diluted PCR product was ftlled in fresh
capillaries and a melting point analysis was performed {Fig. 6}. The correct genotype was only obtained with a 1: 10 dilution of the PCR product.
In order to check the influence of the probe composition, a second set of
hybridization probes {probe set 2} was designed {Table 1; Fig. I}. This alternative
probe system produced identical results in the melting point analysis of the heterozygous sample, i.e., only asymmetric PCR revealed the correct genotype {data
not shown}. The results were also reproduced when a different primer set {Table 1;
primer set 2} was combined with hybridization probe system 1 (data not shown).
In order to prove the reliability of the assay, nine different samples were genotyped
with the first set of probes and asymmetric amplification conditions (Fig. 7). The
result was identical to that of an RFLP analysis.