Typing of multiple single-nucleotide polymorphisms using ribonuclease cleavage of DNA/RNA chimeric single-base extension primers and detection by MALDI-TOF mass spectrometry

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 -