A novel MALDI-TOF based methodology for genotyping single nucleotide polymorphisms

Abstract

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.

Figure 1

Marker TSC0033440 (G/C polymorphism) genotyped without ddNTPs, using Thermosequenase, on three DNA samples representing each of the three possible genotypes. Forward informative primer (5663 Da) was used for the primer extension with 12.5 µM dNTP mix depleted of dGTP. The expected primer extension products were 5966 Da (pT extension) and 6559 Da (pTpCpT extension) for the G and C alleles, respectively. (A) Homozygous C/C sample for TSC0033440. (B) Heterozygous G/C sample for TSC0033440. (C) Homozygous sample G/G for TSC0033440.

Figure 2

Marker TSC0033440 (G/C polymorphism). Exo I- and SAP-treated PCR products, genotyped without ddNTPs, re-using AmpliTaq Gold, from the PCR step on three pre-genotyped DNA samples. Reverse informative primer (5622 Da) was used for the primer extension with 25 µM dNTP depleted of dGTP. The expected extension products were 5936 Da (pA extension) and 6538 Da (pApCpA extension) for the C and G alleles, respectively. (A) Homozygous C/C sample for TSC0033440. (B) Heterozygous G/C sample for TSC0033440. (C) Homozygous G/G sample for TSC0033440.

Figure 3

Schematic picture of the NUDGE process. In this example, starting heterozygote target was G/A polymorphism for a given marker. (1) PCR with informative forward primer twice the concentration of the reverse primer resulting in accumulation of small PCR products. (2) Degradation of excess dNTPs using SAP. (3) Genotyping with NUDGE using the existing forward informative primer and dGTP depleted dNTP mixture. (4) Purification of the accumulated extension products to be analyzed by MALDI–TOF mass spectrometry.

Figure 4

Markers TSC0834913 and TSC0920615 genotyped together (duplex) using the NUDGE protocol and dGTP/dATP depleted mixture. (A) Sample A was homozygous for both markers. TSC0834913 info_Fwd (6412 Da) and A allele (6716 Da), TSC0920615 info_Rev (5541 Da) and T allele (5830 Da). (B) Sample B was homozygous for TSC0834913 A allele (6716 Da) and TSC0920615 homozygous for the G allele (6119 Da); the asterisk-labeled peak had a mass of 5845 Da and did not match the T allele and was excluded in the calibration process. (C) Sample C was heterozygous for the TSC0834913 A (6716 Da) and C alleles (7006 Da) and also for TSC0920615 G (6119 Da) and T alleles (5830 Da). The extra peaks labeled with squares are from the TSC0834913 NUDGE_Info_Rev primer (7026 Da) and its extensions that also interrogate the SNP C (7315 Da) and A alleles (7619 Da). The extra peaks labeled with diamonds are extensions from the TSC0920615 NUDGE_Info_Fwd (4824 Da) primer that also interrogates the SNP T (5114 Da) and G alleles (5418 Da). Since the TSC0834913 NUDGE_Info_Rev and TSC0920615 NUDGE_Info_Fwd were in lower concentration than the other informative primers, the peaks are of lower intensity, but they can be used to confirm the data.