Typing of Multiple Single-Nucleotide Polymorphisms Using Ribonuclease Cleavage of DNA/RNA Chimeric Single-Base Extension Primers and Detection by MALDI-TOF Mass Spectrometry

2005 ◽  
Vol 77 (16) ◽  
pp. 5229-5235 ◽  
Author(s):  
J. Mengel-Jørgensen ◽  
J. J. Sanchez ◽  
C. Børsting ◽  
F. Kirpekar ◽  
N. Morling
Keyword(s):  
Mass Spectrometry ◽  
Single Nucleotide Polymorphisms ◽  
Nucleotide Polymorphisms ◽  
Single Base Extension ◽  
Single Nucleotide ◽  
Single Base ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof ◽  
Base Extension

scholarly journals Multiplex Genotyping of Cytochrome P450 Single-Nucleotide Polymorphisms by Use of MALDI-TOF Mass Spectrometry

2007 ◽  
Vol 53 (5) ◽  
pp. 933-939 ◽  
Author(s):  
Ashish Misra ◽  
Jun-Yan Hong ◽  
Sobin Kim
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Single Nucleotide Polymorphisms ◽  
Solid Phase ◽  
Affinity Purification ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof ◽  
Base Extension

Abstract Background: Polymorphisms in cytochrome P450 (CYP450) genes contribute to interindividual differences in the metabolism of xenobiotic chemicals, including the vast majority of drugs, and may lead to toxicity and adverse drug reactions. Studies on these polymorphisms in research and diagnostic settings typically involve large-scale genotyping and hence require high-throughput assays. Methods: We used the previously developed solid-phase capture–single-base extension (SPC-SBE) approach for concurrent analysis of 40 single-nucleotide polymorphisms (SNPs) of CYP2C9 and 50 SNPs of CYP2A13, both genes belonging to the CYP450 family. Desired SNP-containing regions for each gene were amplified in a single-step multiplex PCR. We designed a library of primers to anneal immediately upstream of the selected SNPs and extended it with biotinylated terminators using PCR products as templates. Biotinylated extension products were isolated by affinity purification and analyzed with MALDI-TOF mass spectrometry to determine SNP genotypes. Results: We analyzed 11 samples for CYP2C9 and 14 samples for CYP2A13 with unambiguous detection of SNPs in all samples. Many samples showed a high occurrence of heterozygotes for both genes, with as many as 10 of 50 SNPs appearing as heterozygotes in 1 sample genotyped for CYP2A13. Conclusions: The SPC-SBE method provides an efficient means for genotyping SNPs from the CYP450 family. This approach is suitable for automation and can be extended to other genotyping applications.

scholarly journals Accurate Single-Nucleotide Polymorphism Allele Assignment in Trisomic or Duplicated Regions by Using a Single Base–Extension Assay with MALDI-TOF Mass Spectrometry

2011 ◽  
Vol 57 (8) ◽  
pp. 1188-1195 ◽  
Author(s):  
Anne L Trewick ◽  
Julia S El-Sayed Moustafa ◽  
Adam J de Smith ◽  
Philippe Froguel ◽  
Gottfried Greve ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Down Syndrome ◽  
Peak Height ◽  
Z Score ◽  
Single Base Extension ◽  
Single Nucleotide ◽  
Single Base ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof ◽  
Base Extension

BACKGROUND The accurate assignment of alleles embedded within trisomic or duplicated regions is an essential prerequisite for assessing the combined effects of single-nucleotide polymorphisms (SNPs) and genomic copy number. Such an integrated analysis is challenging because heterozygotes for such a SNP may be one of 2 genotypes—AAB or ABB. Established methods for SNP genotyping, however, can have difficulty discriminating between the 2 heterozygous trisomic genotypes. We developed a method for assigning heterozygous trisomic genotypes that uses the ratio of the height of the 2 allele peaks obtained by mass spectrometry after a single-base extension assay. METHODS Eighteen COL6A2 (collagen, type VI, alpha 2) SNPs were analyzed in euploid and trisomic individuals by means of a multiplexed single-base extension assay that generated allele-specific oligonucleotides of differing Mr values for detection by MALDI-TOF mass spectrometry. Reference data (mean and SD) for the allele peak height ratios were determined from heterozygous euploid samples. The heterozygous trisomic genotypes were assigned by calculating the z score for each trisomic allele peak height ratio and by considering the sign (+/−) of the z score. RESULTS Heterozygous trisomic genotypes were assigned in 96.1% (range, 89.9%–100%) of the samples for each SNP analyzed. The genotypes obtained were reproduced in 95 (97.5%) of 97 loci retested in a second assay. Subsequently, the origin of nondisjunction was determined in 108 (82%) of 132 family trios with a Down syndrome child. CONCLUSIONS This approach enabled reliable genotyping of heterozygous trisomic samples and the determination of the origin of nondisjunction in Down syndrome family trios.

Single-Base Extension and ELISA-Based Approach for Single-Nucleotide Polymorphisms Genotyping

2010 ◽  
Vol 163 (5) ◽  
pp. 573-576 ◽  
Author(s):  
Guibo Liu ◽  
Yongxia Cheng ◽  
Wei Zhao ◽  
Zaishun Jin ◽  
Hongbo Shan ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Nucleotide Polymorphisms ◽  
Single Base Extension ◽  
Single Nucleotide ◽  
Single Base ◽  
Base Extension
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