Introduction

Rapid discoveries of the genetic causes of human diseases are pointing to multiple mutations either in a single gene or in multiple genes. Multiplexing technologies that allow simultaneous detection of several mutations have been developed to increase power and decrease cost associated with genetics tests. On one hand, high-throughput multiplexing approaches such as chips and tandem mass spectrometry can interrogate multiple loci (>100) and are well suited for the research arena. Lower throughput techniques that interrogate several genes and mutations (<100) are now being integrated with success in clinical laboratories (as exemplified by the pharmacogenetics tests on the CYP genes family). In many cases, the analysis of only a few mutations is medically necessary, and simplicity and cost of the assay drive the choice of technology. Fluorescence monitoring of sequence-specific probes has been classically used to genotype a single locus. Analysis of several loci is possible by combining different fluorophores and temperatures, but this usually requires as many probes as loci (1,2,3). We have recently described a probe design that allows simultaneous analysis of several loci too far apart to be interrogated by a single continuous probe (4). Because these probes span two or more noncontiguous regions of DNA or loci, we refer to them as loci-spanning probes or LSProbes. In the probe, template sequences present between the loci are omitted, and formation of a template bulge allows continuous binding of the probe (Figure 1). We have demonstrated that the stability of LSProbes depends on binding on each side of the template bulge and that the probe dissociates as a unit allowing direct molecular haplo-typing of several loci (4). Here we describe a one-step genotyping assay for three mutations of the β-globin gene with LSProbes that relies on fluorescence resonance energy transfer (FRET) (Figure 1A) or fluorescent double-stranded DNA specific dyes (5) for detection (Figure 1B).

Figure 1. Loci-scanning probe (LSProbe) design. (Click to enlarge)

Mutations in the β-globin gene are responsible for various hemoglobinopathies (6) and have been targeted for diagnostics and screening purposes. The three common hemoglobin variants, HbS (sickle cell anemia), HbC, and HbE are due to mutations in close proximity. Several multiplex technologies previously described use separate probes for the HbS/C and the HbE loci (2,3,7,8). Here we describe LSProbes that analyze simultaneously all three loci.

Materials and Methods

Samples

Unidentified DNA samples with established genotypes (2,8) were used. They had been extracted from whole blood using the MagNA Pure LC instrument (Roche Diagnostics, Indianapolis, IN, USA) or from blood spots (2).

PCR and Melting Analysis

For analysis of the FRET-LSProbe system, PCRs were performed on the LightCycler® instrument (Roche Diagnostics) using the following conditions: approximately 50 ng DNA were used as template in a reaction containing 0.1 µM forward (5′-AGTCAGGGCAGAGCCATCTA-3′), 0.5 µM reverse (5′-GTTTC TATTGGTCTCCTTAAACCTG-3′) PCR primers, and 0.2 µM LSProbe (sequence presented in Table 1) and the anchor hybridization probe (5′-LC640-GTGCACCATGGTGTCTGT TTG AGGTTGCTAGTG A AC A- C 3-3′ blocker; Idaho Technology, Salt Lake City, Utah, USA) in 1× LightCycler DNA Master Hybridization probes (Roche Diagnostics) adjusted to 3 mM final concentration of MgCl₂. The following conditions were used for the reactions: denaturation at 94°C for 0 s, annealing at 63°C for 30 s, and extension at 75°C for 0 s for 40 cycles. Programmed transition rates were 20°C/s. The amplification cycles were followed by a melting cycle as follows: 94°C with 5 s holding time, cooled to 30°C, and held for 20 s. Temperature was then raised to 65°C with a transition rate of 0.3°C/s. Fluorescence was continuously monitored during the melt. Melting curves were converted into negative derivative curves of fluorescence with respect to temperature (-dF/dT) by the LightCycler data analysis software version 3.5 (Roche Diagnostics).

Table 1. Sequence of LSProbes and Templates (Click to enlarge)

The PCR using the unlabeled LSProbe was performed as described above, except for the addition of 0.1×LCGreen I (Idaho Technology) and the absence of the anchor probe. The sequence of the unlabeled LSProbe is identical as the FRET-LSProbe (Table 1). The amplification was as described above, but 60 cycles were performed instead of 40 in order to increase the fluorescence signal detected with 0.1× LCGreen I. The capillaries were analyzed by high resolution melting in an HR-1 instrument (Idaho Technology) using a melting ramp of 0.3°C/s from 35°–65°C. HiR1 v.6g software (Idaho Technology) was used for data normalization and analysis.