Detection and localization of single base changes by denaturing
gradient gel electrophoresis.
Taking into account the time needed to set up and run the PCR and to perform DHPLC analysis, we were able to screen the entire coding region of the ATRX gene in ~16 h compared with 1 week for denaturing
gradient gel electrophoresis analysis.
This map predicts a location where DNA fragments will be denatured and stopped to form a band in a denaturing
gradient gel (Lerman and Silverstein 1987).
Subsequently, 330 amplicons were screened blind with denaturing
MADGE (below) as the analytical platform.
The percentage of substrate converted to product was determined after denaturing
polyacrylamide gel electrophoresis using standard phosphorimager analysis as above.
gradient gel electrophoresis and single-strand conformation polymorphism analysis are more difficult to automate (9).
The PCR product was subjected to heteroduplex induction as follows: preheating at 95 [degrees]C; denaturing
for 5 min at 95 [degrees]C; gradually reannealing from 95 [degrees]C to 10 [degrees]C with 1 [degrees]C/min; and storage at 4 [degrees]C before analysis.
Several methods that accelerate mutational screening before sequencing have been developed, such as single-strand conformation polymorphism analysis (9), heteroduplex gel analysis (10), and denaturing
gradient gel electrophoresis (11,12).
FAM-DYS271 assures that the 364 multichannel fluorescence detector with Varian's Helix System are working at optimal conditions for denaturing
high performance liquid chromatography (DHPLC).
We have chosen denaturing
gradient gel electrophoresis (DGGE) of PCR-amplified material, by which fragments up to 800 by in length can be screened for the presence of mutations.
Recently, a new technique for sensitive, relatively inexpensive and automated high-throughput screening of mutations, denaturing
HPLC (DHPLC), was introduced (10-13).
Products are left without traces of denaturing
solvents or potentially mutagenic chemicals being utilized in current virus inactivation technologies.