SNP genotyping by mass spectrometry using mass-modified nucleotides
Mass spectrometry (MS)-based genotyping enables multiplexed detection of single nucleotide polymorphisms (SNPs) and offers an alternative to fluorescence-based assays[1].
Allelic discrimination is based on single base extension (SBE), where a primer anneals immediately upstream of the SNP site and is extended by one chain-terminating, complementary nucleotide (Fig. 1). MALDI-TOF MS determines the mass of the resulting extension product, allowing precise identification of the sequence and thus SNP alleles.
Key requirements for nucleotides in SNP genotyping by MS include:
- Chain termination (no 3'-OH), preventing further extension (Fig. 1)
- High enzymatic incorporation efficiency
- Introduction of a unique mass tag to each extension product to facilitate robust MS resolution and thus unambiguous allele discrimination
What are SNPs?
„SNPs are single base pair positions in genomic DNA at which different sequence alternatives (alleles) exist in normal individuals in some population(s), wherein the least frequent allele has an abundance of 1% or greater.“[7]Coding-region SNPs may alter protein structure and function, whereas non-coding SNPs can influence transcriptional and post-transcriptional regulation[8]. Both types contribute to interindividual variation in disease susceptibility and drug response, making SNPs valuable markers in human genetics and pharmacogenomics[9,10].
Figure 1: Lack of 3′-OH group is required for chain termination properties.
While Acyclo-nucleoside-triphosphates (Acyclo-NTPs) (A) substitute a 2‐hydroxyethoxymethyl group for the ribose moiety, 2',3'-Dideoxynucleoside-5'-triphosphate (ddNTPs) (B) lack both the 2‘- and 3‘-OH groups. Both core structures prevent further chain extension after nucleotide incorporation.
Table 1: Overview of Acyclo-NTPs and Biotin-labeled ddNTPs
| Products | Cat. No. | Amount | Price |
|---|---|---|---|
| Acylco-ATP | NU-538S | 50 µl (100 mM) | 148,50 € |
| NU-538L | 5 x 50 µl (100 mM) | 445,50 € | |
| NU-538-CSTM | Custom specific bulk amount | Request a Quote | |
| Acyclo-GTP | NU-540S | 50 µl (100 mM) | 148,50 € |
| NU-540L | 5 x 50 µl (100 mM) | 445,50 € | |
| NU-540-CSTM | Custom specific bulk amount | Request a Quote | |
| Acylco-CTP | NU-539S | 50 µl (100 mM) | 148,50 € |
| NU-539L | 5 x 50 µl (100 mM) | 445,50 € | |
| NU-539-CSTM | Custom specific bulk amount | Request a Quote | |
| Acyclo-5-Br-UTP | NU-541S | 50 µl (100 mM) | 148,50 € |
| NU-541L | 5 x 50 µl (100 mM) | 445,50 € | |
| NU-541-CSTM | Custom specific bulk amount | Request a Quote | |
| Biotin-11-ddGTP | NU-1618-BIOX | 25 µl (1 mM) | 403,50 € |
| NU-1618-BIOX-CSTM | Custom specific bulk amount | Request a Quote | |
| Biotin-11-ddATP | NU-1612-BIOX | 25 µl (1 mM) | 355,60 € |
| NU-1612-BIOX-CSTM | Custom specific bulk amount | Request a Quote | |
| Biotin-11-ddCTP | NU-850-BIOX-S | 25 µl (1 mM) | 91,00 € |
| NU-850-BIOX-L | 5 x 25 µl (1 mM) | 333,10 € | |
| NU-850-BIOX-CSTM | Custom specific bulk amount | Request a Quote | |
| Biotin-11-ddUTP | NU-1619-BIOX-S | 25 µl (1 mM) | 86,90 € |
| NU-1619-BIOX-L | 5 x 25 µl (1 mM) | 318,10 € | |
| NU-1619-BIOX-CSTM | Custom specific bulk amount | Request a Quote | |
| Biotin-16-ddUTP | NU-253-BIO16-S | 25 µl (1 mM) | 96,30 € |
| NU-253-BIO16-L | 5 x 25 µl (1 mM) | 352,10 € | |
| NU-253-BIO16-CSTM | Custom specific bulk amount | Request a Quote |
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Selected References:
1] Ross et al. (1998) High level multiplex genotyping by MALDI-TOF mass spectrometry. Nature Biotechnology 16:1347.
[2] Kim et al. (2002) Solid phase capturable dideoxynucleotides for multiplex genotyping using mass spectrometry. Nucleic Acids Res. 15(30):e85.
[3] Smith et al. (2003) Digital genotyping using molecular affinity and mass spectrometry. Nature Reviews Genetics 4:1001.
[4] Mengel-Jørgensen et al. (2004) Multiplex Y chromosome SNP genotyping using MALDI-TOF mass spectrometry. International Congress Series 1261:15.
[5] Di Degusto et al. (2003) Single base extension (SBE) with proofreading polymerases and phosphorothioate primers:improved fidelity in single-substrate assays. Nucleic Acids Res. 31(3):e7.
[6] Gardener et al. (2019) Therminator DNA Polymerase: Modified Nucleotides and Unnatural Substrates. Front. Mol. Biosci. 6(28):doi:10.3389/fmolb.2019.00028.
[7] Brookes (1999) The essence of SNPs. Gene 234 (2):177.
[8] Ward et al. (2012) Interpreting noncoding genetic variation in complex traits and human disease. Nat. Rev. Genet. 13(8):493.
[9] Collins et al. (1997) Variations on a theme: cataloging human DNA sequence variation. Science 278(5343):1580.
[10] Johnson et al. (2001) Pharmacogenomics: impact of polymorphisms on drug response. Annu. Rev. Pharmacol. Toxicol. 41:539.