Analysis of clinically relevant single-nucleotide polymorphisms by use of microelectronic array technology.
BACKGROUND
Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes.


METHODS
Primer pairs, with one containing a 5'-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated. After denaturation, the biotinylated strand was electronically targeted to discrete sites on streptavidin-coated gel pads surfaces by use of a Nanogen Molecular Workstation. Allele-specific dye-labeled oligonucleotide reporters were used for detection of wild-type and variant sequences. Methods were developed for SNPs in genes, including factor VII, beta-globin, and the RET protooncogene. We genotyped 331 samples for five DNA variations in the factor VII gene, >600 samples from patients with beta-thalassemia, and 15 samples for mutations within the RET protooncogene. All samples were previously typed by various methods, including DNA sequence analysis, allele-specific PCR, and/or restriction enzyme digestion of PCR products.


RESULTS
Analysis of amplified DNA required 4-6 h. After mismatched DNA was removed, signal-to-noise ratios were >5. More than 940 samples were typed with the microelectronic array platform, and results were totally concordant with results obtained previously by other genotyping methods.


CONCLUSIONS
The described protocols detect SNPs of clinical interest with results comparable to those of other genotyping methods.
Santacroce
Rosa
R
Department of Pediatrics, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Ratti
Antonia
A
Caroli
Francesco
F
Foglieni
Barbara
B
Ferraris
Alessandro
A
Cremonesi
Laura
L
Margaglione
Maurizio
M
Seri
Marco
M
Ravazzolo
Roberto
R
Restagno
Gabriella
G
Dallapiccola
Bruno
B
Rappaport
Eric
E
Pollak
Eleanor S
ES
Surrey
Saul
S
Ferrari
Maurizio
M
Fortina
Paolo
P
D005167:Factor VII / Q000235:genetics; D005914:Globins / Q000235:genetics; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015513:Oncogene Proteins / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics
D015513:Oncogene Proteins; D005167:Factor VII; D005914:Globins; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D020794:Receptor Protein-Tyrosine Kinases