Real-time PCR genotyping using displacing probes

Abstract

Simple and reliable genotyping technology is a key to success for high-throughput genetic screening in the post-genome era. Here we have developed a new real-time PCR genotyping approach that uses displacement hybridization-based probes: displacing probes. The specificity of displacing probes could be simply assessed through denaturation analysis before genotyping was implemented, and the probes designed with maximal specificity also showed the greatest detection sensitivity. The ease in design, the simple single-dye labeling chemistry and the capability to adopt degenerated negative strands for point mutation genotyping make the displacing probes both cost effective and easy to use. The feasibility of this method was first tested by detecting the C282Y mutation in the human hemochromatosis gene. The robustness of this approach was then validated by simultaneous genotyping of five different types of mutation in the human beta-globin gene. Sixty-two human genomic DNA samples with nine known genotypes were accurately detected, 32 random clinical samples were successfully screened and 114 double-blind DNA samples were all correctly genotyped. The combined merits of reliability, flexibility and simplicity should make this method suitable for routine clinical testing and large-scale genetic screening.

Figure 1

Principle of real-time PCR genotyping using displacing probes. A pair of displacing probes is included in the reaction. In the presence of wild-type templates, only Fam-labeled wild-type probes hybridize to the amplicons and generate green fluorescence (fluorescent cross-annealing), whereas the Texas Red-labeled mutant probes retain their duplex and cannot produce red fluorescence (non-fluorescent self-annealing). In the presence of heterozygous templates, both wild-type and mutant probes hybridize to amplicons and generate both green and red fluorescence. In the presence of mutant templates, only the Texas Red-labeled mutant probes hybridize to the amplicons, generating red fluorescence, whereas Fam-labeled wild-type probes remain dark.

Figure 2

The effect of the length difference between the negative strand and the positive strand (A), and the effect of the length of the probe (B) on the detection sensitivity and specificity of the displacing probe-based real-time PCR. Real-time PCR was carried out using the primer pair for IVS-2–654 site in the HBB gene with both wild-type and mutant templates. Water was used as the blank template. The probes of varied length used were based on the wild-type probe for the IVS-2–654 site. The probe was named according to the numbers of nucleotide of its positive (before) and negative strands (after), which were separated by a slash (e.g. probe 29/28, 5′-Fam-CTGGGTTAAGGCAATAGCAATATCTCTGC-PO₄-3′/5′-CAGAGATATTGCTATTGC CTTAACCCAG-Dabcyl-3′, has a positive strand of 29 nt and a negative strand of 28 nt). The corresponding T_m values of both positive and negative strands are shown separately and are separated by a slash. The length change was made starting from the 3′-end of the positive strand, and accordingly from the 5′-end of the negative strands. Nucleotide changes were based on the original HBB sequence line.

Figure 3

Thermal denaturation curves of the five pairs of displacing probes used for HBB genotyping. The fluorescence intensity of solutions containing wild-type probes (upper panel) and mutant probes (lower panel) was plotted as a function of temperature in the absence of targets (gray solid lines), in the presence of the wild-type target (black solid lines) and in the presence of the mutant targets (dashed lines). The wild-type targets are oligonucleotides perfectly complementary to the positive strand of the wild-type probes, and the mutant targets are oligonucleotides perfectly complementary to the positive strand of the mutant-type probes.

Figure 4

Real-time PCR quantification using displacing probes. PCR was conducted using the primer pair and the wild-type probe for IVS-2–654. Ten-fold serial dilutions of each genomic DNA template, ranging from 7.5 × 10²–7.5 × 10^–3 ng (A, from left to right) per 25-µl reaction and the negative control were amplified. The linear relationship (B) between the number of threshold cycle values and the logarithm of the mass amount of genomic DNA present in a sample demonstrated that quantitative determinations could be made over a target concentration range of at least six orders of magnitude.

Figure 5

(A) Real-time PCR genotyping of HFE. Typical genotyping results were from four PCRs with wild-type (black solid line), mutant type (dashed line), heterozygous templates (dash-dotted line) and negative control (gray solid line) respectively. The color of the fluorescence that developed during PCR identified each of the three genotypes. (B) PCR–RFLP results. The upper panel showed the restriction sites located in the wild-type amplicon (282Cys) and mutant amplicon (282Tyr). The lower panel shows the electrophoresis results of different amplicons treated or not treated with RsaI. The wild-type PCR product (lane 2) was cleaved into two fragments, 250 and 140 bp; the mutant product (lane 3) was cut to three fragments, 250, 111 and 29 bp (note: the 29 bp fragment band is hardly visualized in this figure); and the heterozygous product (lane 4) was digested into four fragments. Lanes 2′, 3′, and 4′ showed the corresponding undigested product of the wild-type, mutant and heterozygous type, all of which were 390 bp. Lane 1 and 1′ were pUC19-MspI (HapII) markers.

Figure 6

Simultaneous real-time PCR genotyping of HBB gene containing five mutations. Fluorescence was recorded at both Fam and Texas Red channels corresponding to the wild-type and mutant probes, respectively. For each mutation, typical three PCR results were shown corresponding to the three genotypes of the templates, they were wild-types (black solid lines), mutant types (dashed lines) and heterozygotes (dash-dotted lines). Templates used were artificial plasmid DNA. Negative control is shown as a gray solid line.

Figure 7

Simultaneous genotyping results of HBB gene from 62 human genomic DNA samples. Human genomic DNA samples of known genotype were listed on the right. The results from each sample were plotted at coordinates that correspond to the threshold cycle for the signal in either wild-type (Fam) or mutant probe (Texas Red) (left panel). For the wild-type samples, only one threshold value was plotted for each sample as the five PCRs gave similar results. In the mutant and heterozygous group, mutation types were illustrated with different colors. Note that the three major genotypes were clearly separated into three groups. The 20 wild-type samples were clustered in the group located in the lower right corner. The 11 homozygous mutant samples were clustered in the group located in the upper left corner, including three samples of CD17/CD17 (blue), five samples of CDs41–42/CDs41–42 (red) and three samples of IVS-2–654/ IVS-2–654 (green). The 31 heterozygous samples were clustered and located in the lower left corner, including six samples of –28(A→G)/N (orange), seven samples of CD17 (A→T)/N (blue), seven samples of CDs41–42 (–TCTT)/N (red), four samples of CDs71–72 (+A)/N (yellow) and seven samples of IVS-2–654 (C→T)/N (green).