Analysis of mutational spectra by denaturing capillary electrophoresis.
The point mutational spectrum over nearly any 75- to 250-bp DNA sequence isolated from cells, tissues or large populations may be discovered using denaturing capillary electrophoresis (DCE). A modification of the standard DCE method that uses cycling temperature (e.g., +/-5 degrees C), CyDCE, permits optimal resolution of mutant sequences using computer-defined target sequences without preliminary optimization experiments. The protocol consists of three steps: computer design of target sequence including polymerase chain reaction (PCR) primers, high-fidelity DNA amplification by PCR and mutant sequence separation by CyDCE and takes about 6 h. DCE and CyDCE have been used to define quantitative point mutational spectra relating to errors of DNA polymerases, human cells in development and carcinogenesis, common gene-disease associations and microbial populations. Detection limits are about 5 x 10(-3) (mutants copies/total copies) but can be as low as 10(-6) (mutants copies/total copies) when DCE is used in combination with fraction collection for mutant enrichment. No other technological approach for unknown mutant detection and enumeration offers the sensitivity, generality and efficiency of the approach described herein.
Ekstrøm
Per O
PO
Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139-4307, USA. peroe@radium.uio.no or peroe@mit.edu
Khrapko
Konstantin
K
Li-Sucholeiki
Xiao-Cheng
XC
Hunter
Ian W
IW
Thilly
William G
WG
D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009691:Nucleic Acid Denaturation / Q000235:genetics
D017931:DNA Primers; D004272:DNA, Mitochondrial