Analysis of Polymerase Chain Reaction Products by Denaturing High-Performance Liquid Chromatography - Clinical Applications of PCR - page 77

Fig. 3. Chromatographic gradient for mutation detection of exon 3 of the
G6PT1 gene.

beads (average diameter 2.3 m), allowing for analysis under a wide range in

pH (2.0-13.0) and temperature (40-80°C) conditions. A positively charged
ion-pairing reagent, TEAA, allows the negatively charged DNA backbone to
interact with the hydrophobic DNAsep cartridge matrix.
1.

µ

Heat the PCR product to 95°C for 3 min and allow it to cool slowly. For an
individual heterozygous for the targeted mutation, the wild-type and mutant DNA
hybridize to form a mixture of hetero- and homoduplexes. This approach is modi-
fied during the analysis of DNA from individuals carrying two identical mutant
alleles (homozygous mutation). The PCR product spanning the homozygous
mutation is mixed with wild-type amplified DNA and hybridized. After this treat-
ment, a sample will contain a mixture of hetero- and homoduplexes. In our case,
we have a homozygous mutation; therefore, in the first part of the analysis, we
analyzed the PCR product amplified from the fetal DNA. In the second part of
the analysis, we mix the PCR product amplified from the fetal DNA with that
amplified from wild-type DNA. The nucleotide sequence of G6PT1 in the wild-
type sample has been confirmed to be normal by direct DNA sequencing.

2. Eluents A (0.1 M TEAA) and B (0.1 M TEAA and 25% ACN) are prepared from

concentrated TEAA (100 mL) ( see Note 5 ).

3. Between 5 and 10 µ L of crude PCR product is loaded on a DNASep column ( see

Note 6 ).

4. The PCR product is eluted from the column by an acetonitrile gradient in

0.1 mol/L TEAA, pH 7.0, at a constant flow rate of 0.9 mL/min ( see Note 7 ). The
gradient is created by mixing eluents A and B. The recommended gradient for
mutation detection is a slope of 2% increase in buffer B per minute ( see Note 8 );
e.g., the gradient for DHPLC analysis of exon 3 of the G6PT1 gene is depicted in
Table 1 and Fig. 3 .

5. Eluted DNA fragments are detected with ultraviolet absorption at wavelength

260 nm by the detector within the DHPLC analyzer ( see Note 9 ).