Single nucleotide polymorphism genotyping by two colour melting curve analysis using the MGB Eclipse Probe System in challenging sequence environment

Abstract

Probe and primer design for single nucleotide polymorphism (SNP) detection can be very challenging for A-T DNA-rich targets, requiring long sequences with lower specificity and stability, while G-C-rich DNA targets present limited design options to lower GC-content sequences only. We have developed the MGB Eclipse Probe System, which is composed of the following elements: MGB Eclipse probes and primers, specially developed software for the design of probes and primers, a unique set of modified bases and a Microsoft Excel macro for automated genotyping, which ably solves, in large part, this challenge. Fluorogenic MGB Eclipse probes are modified oligonucleotides containing covalently attached duplex-stabilising dihydrocyclopyrroloindole tripeptide (DPI3), the MGB ligand (MGB is a trademark of Epoch Biosciences, Bothell, WA), which has the combined properties of allowing the use of short sequences and providing great mismatch discrimination. The MGB moiety prevents probe degradation during polymerase chain reaction (PCR), allowing the researcher to use real time data; alternatively, hybridisation can be accurately measured by a post-PCR two-colour melt curve analysis. Using MGB Eclipse probes and primers containing modified bases further enhances the analysis of difficult SNP targets. G- or C-rich sequences can be refractory to analysis due to Hoogsteen base pairing. Substitution of normal G with Epoch's modified G prevents Hoogsteen base pairing, allowing both superior PCR and probe-based analysis of GC-rich targets. The use of modified A and T bases allows better stabilisation by significantly increasing the Tm of the oligonucleotides. Modified A creates A-T base pairs that have a stability slightly lower than a G-C base pair, and modified T creates T-A base pairs that have a stability about 30 per cent higher than the unmodified base pair. Together, the modified bases permit the use of short probes, providing good mismatch discrimination and primers that allow PCR of refractory targets. The combination of MGB Eclipse probes and primers enriched with the MGB ligand and modified bases has allowed the analysis of refractory SNPs, where other methods have failed.

Figure 1

a) Mechanism of MGB Eclipse probe detection. P is a polymerase, F is a fluorophore and Q is the Eclipse™ Dark Quencher; b) The structure of an MGB Eclipse probe; c) to e) are the structures of modified G, A and T, respectively. dR is 2'-deoxyribofuranoside, R₁ and R₂ are proprietary substituents.

Figure 2

Partial sequence of the selectin E gene (gi:4506870; rs3917410 (SELE-02). The SNP is shown in square brackets. The primer and probe sequences are underlined and shown below. Q is the Eclipse Dark Quencher. The alleles are in bold and underlined in the probe sequences. 'g' is modified G. Probes are complementary to the shown sequence. Fa and Fb are fluorescein and tetrachlorofluorescein, respectively.

Figure 3

Genotyping of SELE-02 alleles using the MGB Eclipse System of 102 CEPH samples. Real-time curves in the fluorescein channels (Fa) and tetrachlorofluorescein (Fb) are shown in a) and c), respectively. Melt curve analysis in the fluorescein and tetrachlorofluorescein channels are shown in b) and d), respectively. A scatter plot of the real-time data from a) and c) is shown in e). Samples 1 and 2 are outlier samples in the scatter plot, genotyped in b) and d) as unequivocally homogeneous wild-type samples. The x-axis and y-axis in a) and c) represent the cycle number and fluorescent signal in fluorescent units, respectively. The x-axis and y-axis in b) and d) represent the temperature in °C and the first derivative, respectively.

Figure 4

A portion of the NAT-I sequence (gi:27754152). The two SNPs are shown in square brackets. The underlined primer sequences are the same for both SNPs and are shown below with corresponding probes. Small letters represent modified bases. Design was made for the antisense strand. Alleles are bold in the probe sequences.

Figure 5

Genotyping of NATI SNPs using the MGB Eclipse System. Melt curves analysis of a) A-T alleles and b) A/C alleles. Fa is fluorescein and Fb is tetrachlorofluorescein. Alleles are underlined in the probe sequences. Small case letters represent modified bases. The x-axis and y-axis represent the temperature in °C and the first derivative, respectively.

Figure 6

a) A portion of the sequence of the RAD23B-02 SNP (gi:12871592). The SNP is shown in parenthesis. Primer sequences are underlined. b) and c) MGB Eclipse data with two probes labelled with fluorescein specific for the A-allele and 5^'-tetrachloro-fluorescein specific for the G-allele, respectively. The alleles are underlined in the probe sequences. 'g' is modified G. The x-axis and y-axis represent the temperature in °C and the first derivative, respectively.

Figure 7

Semi-automated genotyping using the MGB Eclipse Melt Macro and genotyping of NAT-1 A/C alleles. a) Shows the melting curves in the fluorescein channel and b) shows the melting curves in the tetrachlorofluorescein channel. The vertical lines z₁ and z₂ are manually set for the T_ms for the A and C alleles, respectively. Similarly, the thresholds in each channel are manually set and are respectively set as double horizontal lines y₁ and y₂ in the fluorescein and tetrachlorofluorescein channels, respectively. c) A subset of automated genotyped alleles from a 384-well plate is listed as an example. Sample in well A7 is no template control (NTC); samples in other wells contain different human genomic DNAs.