Document Detail


Typing of multiple single-nucleotide polymorphisms using ribonuclease cleavage of DNA/RNA chimeric single-base extension primers and detection by MALDI-TOF mass spectrometry.
MedLine Citation:
PMID:  16097763     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
A novel single-base extension (SBE) assay using cleavable and noncleavable SBE primers in the same reaction mix is described. The cleavable SBE primers consisted of deoxyribonucleotides and one ribonucleotide (hereafter denoted chimeric primers), whereas the noncleavable SBE primers consisted of only deoxyribonucleotides (hereafter denoted standard primers). Biotin-labeled ddNTPs were used in the SBE reaction, and the SBE products were purified using the monomeric avidin triethylamine purification protocol, ensuring that only primers extended with a biotin-ddNTP in the 3'-end were isolated. A ribonuclease mix was developed to specifically cleave the chimeric primers, irrespective of the base of the ribonucleotide, whereas standard primers without a ribonucleotide were unaffected by the ribonuclease treatment. The SBE products were analyzed in linear mode using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The cleaved SBE products were detected in the 2000-5500 m/z range, and the noncleaved SBE products were detected in the 5500-10 000 m/z range. The method was validated by typing 17 Y chromosome single-nucleotide polymorphisms in 100 males with a 17-plex SBE package containing 9 chimeric primers and 8 standard primers.
Authors:
J Mengel-Jørgensen; J J Sanchez; C Børsting; F Kirpekar; N Morling
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Analytical chemistry     Volume:  77     ISSN:  0003-2700     ISO Abbreviation:  Anal. Chem.     Publication Date:  2005 Aug 
Date Detail:
Created Date:  2005-08-15     Completed Date:  2007-03-15     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  5229-35     Citation Subset:  IM    
Affiliation:
Department of Forensic Genetics, Institute of Forensic Medicine, University of Copenhagen, 11 Frederik V's Vej, DK-2100 Copenhagen Ø, Denmark. jonas.mengel@forensic.ku.dk
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MeSH Terms
Descriptor/Qualifier:
Base Sequence
DNA Primers / chemistry,  genetics*,  metabolism*
Molecular Sequence Data
Polymorphism, Single Nucleotide / genetics*
RNA / chemistry,  genetics*,  metabolism*
Ribonucleases / metabolism*
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*
Chemical
Reg. No./Substance:
0/DNA Primers; 0/RNA primers; 63231-63-0/RNA; EC 3.1.-/Ribonucleases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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