Heteroduplex-based genotyping with microchip electrophoresis and dHPLC.
This work compares the methods of mutation detection via denaturing high-performance liquid chromatography (dHPLC) and a microchip-based heteroduplex analysis (HA) method. The mutations analyzed were 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 with, as additional examples, 188del11 and 5396 + 1G --> A in BRCA1. Our HA method is based upon the use of a replaceable, highly denaturing sieving matrix that has dynamic coating capabilities, rendering our method relatively insensitive to contamination. We have found significant advantages in the microchip analysis in terms of reagent consumption, ease of use, versatility, simplicity of the protocol, the lack of constraints upon sample preparation or content, and the lack of parameters that need be adjusted. Although HA methods have a lower sensitivity than that of dHPLC, the electropherograms of the present HA method appear to provide more information and may allow mutations within the same amplicon to be distinguished. Although the dHPLC method has a remarkably high sensitivity, with this sensitivity there come constraints that may prevent it, in its present form, from being used in some applications, particularly those involving higher levels of integration. The advantages of the present HA method, along with recent developments in microchip-based single-nucleotide polymorphism (SNP) detection and high-throughput arrays, suggest that microchip-based systems could provide compact and integrated platforms capable of large-scale genotyping or mutational screening.
Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta, Canada T6G 2V4.
Tim
T
Footz
Martin J
MJ
Somerville
Robert
R
Tomaszewski
Kathleen A
KA
Sprysak
Christopher J
CJ
Backhouse
D019313:BRCA1 Protein / Q000235:genetics; D024682:BRCA2 Protein / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D004586:Electrophoresis / Q000379:methods*; D005838:Genotype; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012680:Sensitivity and Specificity
D019313:BRCA1 Protein; D024682:BRCA2 Protein