Abstract
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Analytical Chemistry |
| Vol/bind | 77 |
| Udgave nummer | 16 |
| Sider (fra-til) | 5229-35 |
| Antal sider | 6 |
| ISSN | 0003-2700 |
| DOI | |
| Status | Udgivet - 2005 |
Bibliografisk note
Keywords: Base Sequence; DNA Primers; Molecular Sequence Data; Polymorphism, Single Nucleotide; RNA; Ribonucleases; Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationCitationsformater
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I: Analytical Chemistry, Bind 77, Nr. 16, 2005, s. 5229-35.
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review
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TY - JOUR
T1 - Typing of multiple single-nucleotide polymorphisms using ribonuclease cleavage of DNA/RNA chimeric single-base extension primers and detection by MALDI-TOF mass spectrometry
AU - Mengel-From, Jonas
AU - Sanchez Sanchez, Juan Jose
AU - Børsting, Claus
AU - Kirpekar, Finn
AU - Morling, Niels
N1 - Keywords: Base Sequence; DNA Primers; Molecular Sequence Data; Polymorphism, Single Nucleotide; RNA; Ribonucleases; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PY - 2005
Y1 - 2005
N2 - 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.
AB - 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.
U2 - 10.1021/ac0502044
DO - 10.1021/ac0502044
M3 - Journal article
C2 - 16097763
SN - 0003-2700
VL - 77
SP - 5229
EP - 5235
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 16
ER -