A novel MALDI-TOF based methodology for genotyping single nucleotide polymorphisms.
A new MALDI-TOF based detection assay was developed for analysis of single nucleotide polymorphisms (SNPs). It is a significant modification on the classic three-step minisequencing method, which includes a polymerase chain reaction (PCR), removal of excess nucleotides and primers, followed by primer extension in the presence of dideoxynucleotides using modified thermostable DNA polymerase. The key feature of this novel assay is reliance upon deoxynucleotide mixes, lacking one of the nucleotides at the polymorphic position. During primer extension in the presence of depleted nucleotide mixes, standard thermostable DNA polymerases dissociate from the template at positions requiring a depleted nucleotide; this principal was harnessed to create a genotyping assay. The assay design requires a primer- extension primer having its 3'-end one nucleotide upstream from the interrogated site. The assay further utilizes the same DNA polymerase in both PCR and the primer extension step. This not only simplifies the assay but also greatly reduces the cost per genotype compared to minisequencing methodology. We demonstrate accurate genotyping using this methodology for two SNPs run in both singleplex and duplex reactions. We term this assay nucleotide depletion genotyping (NUDGE). Nucleotide depletion genotyping could be extended to other genotyping assays based on primer extension such as detection by gel or capillary electrophoresis.
Blondal
Thorarinn
T
Decode Genetics Inc, Sturlugata 8, 101 Reykjavik, Iceland.
Waage
Benedikt G
BG
Smarason
Sigurdur V
SV
Jonsson
Frosti
F
Fjalldal
Sigridur B
SB
Stefansson
Kari
K
Gulcher
Jeffery
J
Smith
Albert V
AV
D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004795:Enzyme Stability; D005838:Genotype; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000191:economics / Q000379:methods*
D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase