Simple two-color array-based approach for mutation detection.
The ability to analyze multiple polymorphic/mutation sites rapidly and accurately is pivotal in all areas of genetic analysis. We have applied single nucleotide primer extension (SNE) for detection of multiple point mutations in a micro-array format using two-color, fluorescent dye-tagged dideoxynucleoside triphosphate terminators (ddNTPs). The oligonucleotide primer ending one nucleotide short of the mutation site being probed is bound to the slide and single-base extended in place with two different Cy5/Cy3 dye-tagged terminators using solution-phase, locus-specific, single-stranded complementary templates generated by PCR from genomic DNA. The composite fluorescence produced contains peaks of distinct wave lengths corresponding to each Cy dye-tagged terminator incorporated, resulting in a fluorescent 'fingerprint' for each DNA target. DNA polymerase-catalyzed incorporation of Cy dye-tagged dideoxynucleoside triphosphates was dependent on the particular dyes, the specific ddNTP, the DNA target concentration, sequence of the template, on-slide temperature cycling and washing conditions. Results from analysis of mutations in the human hemochromatosis and connexin 26 genes show that this approach has several advantages over existing methods and is simple, rapid, robust, cost effective and accurate with potential applications in many areas of genetic analysis.
Fortina
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P
Department of Pediatrics, University of Pennsylvania School of Medicine and The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. fortina@mail.med.upenn.edu
Delgrosso
K
K
Sakazume
T
T
Santacroce
R
R
Moutereau
S
S
Su
H J
HJ
Graves
D
D
McKenzie
S
S
Surrey
S
S
D019943:Amino Acid Substitution; D002232:Carbocyanines / Q000737:chemistry; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D006680:HLA Antigens / Q000235:genetics; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins; D017354:Point Mutation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017384:Sequence Deletion
D002232:Carbocyanines; D017630:Connexins; D017931:DNA Primers; D015342:DNA Probes; D005456:Fluorescent Dyes; C103673:HFE protein, human; D006680:HLA Antigens; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins; C089187:cyanine dye 3; C085321:cyanine dye 5; D000072259:Connexin 26