Multiplex detection and SNP genotyping in a single fluorescence channel

Abstract

Probe-based PCR is widely used for SNP (single nucleotide polymorphism) genotyping and pathogen nucleic acid detection due to its simplicity, sensitivity and cost-effectiveness. However, the multiplex capability of hydrolysis probe-based PCR is normally limited to one target (pathogen or allele) per fluorescence channel. Current fluorescence PCR machines typically have 4-6 channels. We present a strategy permitting the multiplex detection of multiple targets in a single detection channel. The technique is named Multiplex Probe Amplification (MPA). Polymorphisms of the CYP2C9 gene (cytochrome P450, family 2, subfamily C, polypeptide 9, CYP2C9*2) and human papillomavirus sequences HPV16, 18, 31, 52 and 59 were chosen as model targets for testing MPA. The allele status of the CYP2C9*2 determined by MPA was entirely concordant with the reference TaqMan® SNP Genotyping Assays. The four HPV strain sequences could be independently detected in a single fluorescence detection channel. The results validate the multiplex capacity, the simplicity and accuracy of MPA for SNP genotyping and multiplex detection using different probes labeled with the same fluorophore. The technique offers a new way to multiplex in a single detection channel of a closed-tube PCR.

Figure 1. Nucleic acid probe design and its melting profile.

(A) Probe consists of a target-hybridising oligonucleotide (THO) labeled with a Fluorophore at the 5′ end and a Quencher at the 3′end, and a partially complementary oligonucleotide (PCO) without a label. The hybrid of THO:PCO is named plus probe. The melting curve shows decreased fluorescence emission when temperature rises (left panel); the negative derivative plot of the emission reading versus temperature reveals a positive value of the melting peak (right panel). (B) Minus probe consists of a THO labeled with a Fluorophore at the 5′ end and a Quencher at the 3′end, and a PCO labeled with a Quencher at the 3′ end. The melting curve shows increased fluorescence emission when temperature rises (left panel); the negative derivative plot of the emission reading versus temperature reveals a negative value of the melting peak (right panel).

Figure 2. Design of HPV16 and HPV18 probes and the use of the probes for amplification and melting curve analysis.

(A) Hybrids of THO:PCO are formed through hybridisation of THO and PCO. (B) Melting profile of the mix of HPV16 and HPV18 probes is plotted as the negative derivative of the emission reading versus temperature. (C) is the graphic presentation of the amplification plot of PCR on five 10-fold serial dilutions of HPV16 templates. (D) is the same reaction as (C) showing the melting curve. (E) is the graphic presentation of the amplification plot of PCR on five 10-fold serial dilutions of HPV18 templates. (F) is the same reaction as (E) showing the melting curve. (G) is the graphic presentation of the amplification plot of PCR on five 10-fold serial dilutions of the mix of HPV16 and HPV18 templates. (H) is the same reaction as (G) showing the melting curve.

Figure 3. Plus probes genotyping SNP CYP2C9*2.

(A) is the graphic presentation of the amplification plot of PCR for genotyping SNP CYP2C9*2 on three genomic DNA samples and three negative controls. (B) is melting curves showing one sample is homozygous for G allele. (C) is melting curves showing one sample is heterozygous. (D) is melting curves showing one sample is homozygous for A allele.

Figure 4. Melting curve analysis of the amplification reactions of various HPV targets.

When a HPV target is present in a reaction, its corresponding probe is consumed and, in comparison with the negative control, the appropriate melting peak in the melting profile is decreased or has disappeared. The negative control melting curve is marked in red; the target melting curve is marked in blue. (A) HPV16 is present. (B) HPV31 is present. (C) HPV52 is present. (D) HPV59 is present.