Rapid detection of deletion, insertion, and substitution mutations via heteroduplex analysis using capillary- and microchip-based electrophoresis.
In this report, we explore the potential of capillary and microchip electrophoresis for heteroduplex analysis- (HDA) based mutation detection. Fluorescent dye-labeled primers (6-FAM-tagged) were used to amplify the DNA fragments ranging from 130 to 400 bp. The effects of DNA fragment length, matrix additives, pH, and salt were evaluated for capillary electrophoresis- (CE) and/or microchip electrophoresis-based HDA, using six heterozygous mutations, 185delAG, E1250X (3867GT), R1443G (4446CG), 5382insC, 5677insA in BRCA1, and 6174delT in BRCA2. For this system, the effective fragment size for CE-based HDA was found in the range of 200-300 bp, however, the effective range was 150-260 bp for microchip-based HDA. Sensitivity studies show CE-based HDA could detect a mutated DNA present at only 1%-10% of the total DNA. Discrimination between wild-type and deletion or insertion mutations in BRCA1 and BRCA2 with CE-based HDA could be achieved in <8 min, while the substitution mutations required 14 min of analysis time. For each mutation region, 15 samples were run to confirm the accuracy and reproducibility of the method. Using the method described, two previously reported mutations, E1038G (3232AG, missense) and 4427 C/T (4427CT, polymorphism), were detected in the tested samples and confirmed by DNA sequencing. Translation of the CE-based methodology to the microchip format allowed the analysis time for each mutation to be decreased to 130 sec. Based on the results obtained with this model system, it is possible that CE-based HDA methodologies can be developed and used effectively in genetic testing. The fast separation time and automated operation afforded with CE instrumentation provide a powerful system for screening mutations that include small deletions, insertions, and point mutations. Translation to the microchip platform, especially to a multichannel microchip system, would allow for screening mutations with high throughput.
Tian
H
H
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Brody
L C
LC
Landers
J P
JP
D024682:BRCA2 Protein; D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics; D017931:DNA Primers; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods / Q000706:statistics & numerical data; D005260:Female; D005456:Fluorescent Dyes; D019398:Genes, BRCA1; D005819:Genetic Markers / Q000235:genetics; D006720:Homozygote; D006801:Humans; D016254:Mutagenesis, Insertional / Q000235:genetics*; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis* / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017384:Sequence Deletion / Q000235:genetics*; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics
D024682:BRCA2 Protein; D014408:Biomarkers, Tumor; D017931:DNA Primers; D005456:Fluorescent Dyes; D005819:Genetic Markers; D009363:Neoplasm Proteins; D009692:Nucleic Acid Heteroduplexes; D014157:Transcription Factors