Technology to accelerate pangenomic scanning for unknown point mutations in exonic sequences: cycling temperature capillary electrophoresis (CTCE).
BACKGROUND
Rapid means to discover and enumerate unknown mutations in the exons of human genes on a pangenomic scale are needed to discover the genes carrying inherited risk for common diseases or the genes in which somatic mutations are required for clonal diseases such as atherosclerosis and cancers. The method of constant denaturing capillary electrophoresis (CDCE) permitted sensitive detection and enumeration of unknown point mutations but labor-intensive optimization procedures for each exonic sequence made it impractical for application at a pangenomic scale.


RESULTS
A variant denaturing capillary electrophoresis protocol, cycling temperature capillary electrophoresis (CTCE), has eliminated the need for the laboratory optimization of separation conditions for each target sequence. Here are reported the separation of wild type mutant homoduplexes from wild type/mutant heteroduplexes for 27 randomly chosen target sequences without any laboratory optimization steps. Calculation of the equilibrium melting map of each target sequence attached to a high melting domain (clamp) was sufficient to design the analyte sequence and predict the expected degree of resolution.


CONCLUSIONS
CTCE provides practical means for economical pangenomic detection and enumeration of point mutations in large-scale human case/control cohort studies. We estimate that the combined reagent, instrumentation and labor costs for scanning the approximately 250,000 exons and splice sites of the approximately 25,000 human protein-coding genes using automated CTCE instruments in 100 case cohorts of 10,000 individuals each are now less than U.S. $500 million, less than U.S. $500 per person.
Ekstrøm
Per O
PO
Department of Surgical Oncology, The Norwegian Radium Hospital, Oslo, Norway. peroe@radium.uio.no
Bjørheim
Jens
J
Thilly
William G
WG
D004252:DNA Mutational Analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D005091:Exons; D015894:Genome, Human; D006801:Humans; D009691:Nucleic Acid Denaturation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D013696:Temperature
Methods Mol Biol. 2000;132:365-86
10547847
PLoS Comput Biol. 2007 May;3(5):e93
17511513
Electrophoresis. 2002 May;23(10):1499-511
12116161
Electrophoresis. 2003 Jan;24(1-2):63-9
12652573
Hum Mutat. 2003 May;21(5):455-65
12673787
J Biomol Screen. 2002 Dec;7(6):501-6
14599347
Biopolymers. 1974;13(9):1859-71
4415504
Proc Natl Acad Sci U S A. 1983 Mar;80(6):1579-83
6220406
Nucleic Acids Res. 1994 Feb 11;22(3):364-9
8127674
Mutat Res. 2005 Mar 1;570(2):267-80
15708585
Electrophoresis. 2005 Jun;26(13):2520-30
15934053
Electrophoresis. 2005 Jun;26(13):2531-8
15948210
Phys Rev E Stat Nonlin Soft Matter Phys. 2005 Jun;71(6 Pt 1):061922
16089780
Mutat Res. 2007 Feb 3;615(1-2):28-56
17101154
Hum Mutat. 2002 Apr;19(4):334-42
11933188