Document Detail

Analysis of clinically relevant single-nucleotide polymorphisms by use of microelectronic array technology.
MedLine Citation:
PMID:  12446467     Owner:  NLM     Status:  MEDLINE    
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.
Rosa Santacroce; Antonia Ratti; Francesco Caroli; Barbara Foglieni; Alessandro Ferraris; Laura Cremonesi; Maurizio Margaglione; Marco Seri; Roberto Ravazzolo; Gabriella Restagno; Bruno Dallapiccola; Eric Rappaport; Eleanor S Pollak; Saul Surrey; Maurizio Ferrari; Paolo Fortina
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Clinical chemistry     Volume:  48     ISSN:  0009-9147     ISO Abbreviation:  Clin. Chem.     Publication Date:  2002 Dec 
Date Detail:
Created Date:  2002-11-26     Completed Date:  2002-12-10     Revised Date:  2012-06-04    
Medline Journal Info:
Nlm Unique ID:  9421549     Medline TA:  Clin Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  2124-30     Citation Subset:  IM    
Department of Pediatrics, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
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MeSH Terms
Factor VII / genetics
Globins / genetics
Oligonucleotide Array Sequence Analysis / methods*
Oncogene Proteins / genetics
Polymorphism, Single Nucleotide
Proto-Oncogene Proteins c-ret
Receptor Protein-Tyrosine Kinases / genetics
Grant Support
K08 HL 03661/HL/NHLBI NIH HHS; P60 HL 38632/HL/NHLBI NIH HHS; R21 CA 83220-01A1/CA/NCI NIH HHS
Reg. No./Substance:
0/Oncogene Proteins; 9001-25-6/Factor VII; 9004-22-2/Globins; EC Proteins c-ret; EC protein, human; EC Protein-Tyrosine Kinases

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