Biomedical Engineering Reference
In-Depth Information
Table 1
Mobile Phase Conditions for Gradient Elution
Step
Time
%A
%B
mL/min
At detector
Loading
0.00
52
48
0.9
2.00
Start gradient
0.50
47
53
2.50
Stop gradient
5.00
38
62
7.00
Start clean
5.10
0
100
7.10
Stop clean
5.60
0
100
7.60
Start equilibrate
5.70
52
48
7.70
Stop equilibrate
8.20
52
48
10.20
6.
A final increase in organic mobile phase cleans the DNASep cartridge matrix,
removing DNA and other contaminants and preparing the DNASep Cartridge for
the next analysis (
see
Notes 10-12
).
3.3. Interpretation
In the sample with only the PCR products amplified from the fetal DNA, a
single peak is observed in the chromatogram (
Fig. 4
). In the sample with PCR
products amplified from the wild-type DNA mixed with that amplified from
the fetal DNA, two peaks are observed (
Fig. 5
). These two findings together
indicate that the patient is homozygous for the G149E mutation. The fetus is
predicted to be affected with GSD Ib. On direct DNA sequencing, a homozy-
gous 1563G>A is found in the fetal DNA (not shown). This finding confirms
the DHPLC results (
see
Notes 13
and
14
).
4. Notes
1.
The website
www.MutationDiscovery.com,
hosted by Transgenomic, provides
researchers access to experimental methods, methods for DHPLC analysis on a
specific gene, and publications on DHPLC analysis of the gene. More than 350
human genes have been screened entirely or partly by DHPLC.
2.
Use metal-free DNA polymerase buffers, such as
Taq
Gold buffer or Optimase
polymerase buffer.
3.
Do not use mineral oil for topping the reaction mixture.
4.
Use no more than 100 ng DNA for the PCR reaction. Otherwise, the system will
quickly be contaminated by injected DNA.
5.
Do not use autoclaved water. The autoclaving process can introduce metal and
other ionic contaminates, which would bind and cause damages to the column.
6.
For quality control of DHPLC analysis, we run control samples before and after
each DHPLC analysis. DHPLC users can try to reduce the cost of analysis by
replacing the commercial mutation standards with in-house validated controls.
