Anal. Chem. 2005, 77, 5229-5235
Typing of Multiple Single-Nucleotide Polymorphisms Using Ribonuclease Cleavage of DNA/RNA Chimeric Single-Base Extension Primers and Detection by MALDI-TOF Mass Spectrometry J. Mengel-Jørgensen,*,† J. J. Sanchez,† C. Børsting,† F. Kirpekar,‡ and N. Morling†
Department of Forensic Genetics, Institute of Forensic Medicine, University of Copenhagen, 11 Frederik V’s Vej, DK-2100 Copenhagen Ø, Denmark, and Department of Biochemistry and Molecular Biology, University of Southern Denmark, 55 Campusvej, DK-5230 Odense M, Denmark
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 timeof-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 550010 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. Single-nucleotide polymorphisms (SNPs) are the most frequent variations in the genome, and a single SNP or a group of SNP loci may hold essential information concerning the health, the effectiveness of drug treatment, or the origin of an individual. The number of interesting SNPs in many diagnostic investigations is typically between 5 and 50, and in such cases, analysis of all SNPs in a single multiplex reaction is a real possibility. Multiplexing is an efficient way to reduce costs, simplify data analysis, and reduce the risk of sample or data mixups. Furthermore, multiplexing is an efficient way to preserve the original sample material, which can be of crucial importance in medical, anthropological, or forensic genetic investigations. Most SNP typing assays are based * Corresponding author. Phone: +45 3532 6266. Fax: +45 3532 6120. E-mail:
[email protected]. † University of Copenhagen. ‡ University of Southern Denmark. 10.1021/ac0502044 CCC: $30.25 Published on Web 07/08/2005
© 2005 American Chemical Society
on single-base extension (SBE) where a primer is hybridized to the target DNA immediately upstream of the SNP and extended by a dideoxyribonucleotide. SBE reactions can be multiplexed, and the SBE products can be detected in a single experiment. One of the most reliable detection platforms is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). However, a recurrent problem for the various MALDI-TOF-based assays has been the detection of A/T SNPs, because it is difficult to reliably separate two extended SBE primers, with masses of minimum 5000 Da, when the mass difference is only 9 Da.1-3 A/T SNPs can be typed using mixtures of dNTPs and ddNTPs (minisequencing), resulting in products that are easily separated because they differ by at least one nucleotide.4 In addition, three diverse SBE assays were developed with the purpose of analyzing extended SBE primers in the 10003000 m/z range, where the resolution of peaks is sufficiently good to separate A/T SNPs.5-8 The SBE primers were either cleaved or partially digested after the SBE reaction, resulting in short SBE products, 3-10 nucleotides in length. The assays mentioned above have all been successfully implemented in the analysis of small multiplexes (
