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Rapid ß-Globin Genotyping by Multiplexing Probe Melting Temperature and Color

¹ Department of Pathology, University of Utah, 50 N. Medical Dr., Salt Lake City, UT 84132;
² Neo Gen Screening, Pittsburgh, PA 15220;
^a author for correspondence: fax 801-581-4517, e-mail Carl_Wittwer{at}hlthsci.med.utah.edu

Single-nucleotide polymorphisms have been identified by many different experimental approaches. The human ß-globin gene has been genotyped by several methods, including PCR followed by restriction digestion (1)(2), denaturing gradient gel electrophoresis (3), allele-specific amplification during PCR, and the ligase chain reaction (4). These methods require several hours and sometimes days for diagnosis. Recently, rapid-cycle PCR has been combined with real-time fluorescence monitoring to detect mutations by fluorescent probe melting point analysis for homogeneous genotyping in <1 h. Fluorescent melting point analysis is a technique that detects mutations by differences in the melting temperature (T_m) of fluorescent oligonucleotides hybridized to different alleles (5)(6)(7). Probes of a single color are usually used for genotyping. Four alleles at two different loci have been genotyped by multiplexing probe T_ms of a single color (8). However, there is a limit to how many alleles can be distinguished by differences in T_m. The ability to use multiple colored probes along with T_m would greatly extend the power of monitoring PCR with fluorescence by allowing greater numbers of loci to be screened for mutations in one reaction. Exon 1 of the ß-globin gene has >50 mutations, which produce various hemoglobinopathies (9). Hemoglobins S, C, and E are common and are routinely screened. Hemoglobin C (Hb C) results from a G-to-A mutation in the first nucleotide of codon 6, whereas hemoglobin S (Hb S) arises from an A-to-T mutation in the second nucleotide of this codon. Hemoglobin E (Hb E) results from a G-to-A mutation in the first nucleotide of codon 26. The close proximity of these three mutations allowed us to design a probe system that discriminated all genotypes using T_m and two probe colors.

The human ß-globin gene sequence (GenBank accession no. U01317) was used to design primers and probes for the amplification of a 214-bp segment containing exon 1 (Fig. 1 a). Because of the high homology between the ß-globin and -globin sequences, the primers (sense, 5'-GTCAGGGCAGAGCCATCTA-3'; antisense, 5'-GTTCTATTGGTCTCCTTAAAGGTG-3') were designed with 3' and additional mismatches to -globin. In addition, because of the close proximity of the hemoglobin mutations, a unique combination of probes was designed to detect Hb S, C, and E alleles. Two probes were labeled with acceptor fluorophores, LightCycler Red 640 (LC Red 640) and LightCycler Red 705 (LC Red 705; Roche Molecular Biochemicals), as mutation detection probes. The third probe was a dual-labeled fluorescein donor probe that spanned the distance between the mutation detection probes. When annealed, resonance energy was transferred from each fluorescein label to either the LC Red 640- or the LC Red 705-labeled probes. The codon 6 detection probe (5'-CTCCTGTGGAGAAGTCTGC-LC Red 640) completely matched the Hb S allele antisense strand. The codon 26 probe (LC Red 705-GTTGGTGGTAAGGCCCTGGT-phosphate) completely matched the Hb E allele antisense strand. Both the LC Red 640 and LC Red 705 probes were obtained from Idaho Technology Biochem. The fluorescein-labeled probe was labeled with two fluorescein (F) molecules attached to the 5' and 3' ends (F-GTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGA-F; Operon). Fifty-five blinded samples of human genomic DNA were randomly selected from samples submitted to Neo Gen Screening for sickle cell hemoglobinopathy screening. The DNA (80–130 ng) was prepared from blots on filter paper (10) and had been genotyped previously by allele-specific cleavage and gel electrophoresis (2).

View larger version (19K): [in this window] [in a new window]   Figure 1. ß-Globin genotyping schematic (a) and -dF/dT derivative melting curves for each detection probe (b). (a), a 214-bp fragment of the ß-globin gene is illustrated. The sense and antisense DNA strands are shown with exon 1 centrally located. Arrows indicate forward (FWD) and reverse (RVS) primers. Mutation probes are indicated as Probe 1 (LC Red 640 labeled) for codon 6 and Probe 2 (LC Red 705 labeled) for codon 26. The dual-labeled fluorescein probe (Probe 3) is positioned between probes 1 and 2. Point mutations are indicated by bold capitals and duplex mismatches by subscripts. (b), panels A–C contain -dF/dT derivative melting curves for genotypes detected by the codon 6 probe labeled with LC Red 640. Panel A shows wild type (——–), homozygous Hb S (- · -), and S-trait (- - - -) -dF/dT melting curves. Panel B shows -dF/dT melting curves of wild type (——–), homozygous Hb C (- · · -), and C-trait (- - - -). Panel C shows -dF/dT melting curves of compound heterozygote Hb S/C (· · · · ·) in comparison with homozygous Hb S (- · -), and Hb C (- · · -). Panel D contains -dF/dT derivative melting curves for genotypes detected by the codon 26 probe labeled with LC Red 705: wild type (——–), homozygous Hb E (- · -), and E-trait (· · · ·). A no-template control (- -) is present in all panels.

PCR and melting curve analysis were preformed on the LightCycler^TM (Roche Molecular Biochemicals). Asymmetric amplification of the antisense strand occurred in 10 µL of 50 mmol/L Tris, pH 8.5 (25 °C), 3 mmol/L MgCl₂, 500 mg/L bovine serum albumin, 0.2 mmol/L each deoxyribonucleoside triphosphate, 0.5 µmol/L sense primer and 1.0 µmol/L antisense primer, 1 U of KlenTaq DNA polymerase (AB Peptides), 0.2 µmol/L LC Red 640 probe, 0.2 µmol/L LC Red 705 probe, 0.4 µmol/L dual fluorescein-labeled probe, and 2.0 µL of human genomic DNA. The samples were thermally cycled 40 times with three temperature segments. The first segment was 94 °C for 0 s at 20 °C/s for denaturation. A second segment of 63 °C for 30 s at 20 °C/s allowed for both primer and probe annealing. A third temperature segment for extension consisted of a temperature ramp at 1 °C/s from 63 to 75 °C. After amplification, the temperature was raised to 94 °C for 5 s, lowered to 35 °C at 1 °C/s, and held at 35 °C for 20 s. Melting curve profiles where obtained by raising the temperature to 80 °C at 0.1 °C/s while collecting fluorescence data continuously. Genotyping of the samples by T_m was accomplished after color compensation of each channel. T_ms were determined by converting melting curves to -dF/dT derivative peaks and fitting the peaks to gaussian curves (LightCycler software; Roche Molecular Biochemicals).

Melting curve analysis of codon 6 (Hb S and Hb C), and codon 26 (Hb E) point mutations is shown in Fig. 1b . Panels A-C of Fig. 1b show derivative plots that display "melting peaks" for the LC Red 640-labeled probe, and panel D displays results from the LC Red 705 probe. Panel A of Fig. 1b shows single peaks for both the homozygous Hb S (T_m = 63.7 °C; SD = 0.18 °C; n = 25) and the wild-type (T_m = 56.4 °C; SD = 0.26 °C; n = 49) genotypes. Two melting peaks are present for the S-trait corresponding to each allele. Panel B of Fig. 1b shows the melting peaks for homozygous Hb C (T_m = 50.9 °C; SD = 0.48 °C; n = 8). The C-trait has both the Hb C and wild-type peaks. The genotype of a compound heterozygote, Hb S/C, is shown in Fig. 1b , panel C, along with the corresponding Hb C and Hb S genotypes. Genotyping of the Hb E allele is shown in fig. 1b , panel D. Single melting peaks are shown for homozygous Hb E (T_m = 66.1 °C; SD = 0.13 °C; n = 3) and wild-type (T_m = 57.1 °C; SD = 0.20 °C; n = 52) genotypes, whereas the E-trait has two melting peaks corresponding to each allele. The codon 6 and codon 26 probes discriminated the 10 most common hemoglobin genotypes in exon 1. Homozygous samples for the wild type (codon 6, n = 21; codon 26, n = 50), Hb S (n = 1), Hb C (n = 2), and Hb E (n = 1) as well as S-trait (n = 23), C-trait (n = 5), E-trait (n = 2), and the compound heterozygotes Hb S/E (n = 1), C/E (n = 1), and S/C (n = 1) were correctly genotyped. The codon 6 probe sequence that showed the best resolution between the wild-type, Hb S, and Hb C alleles was completely matched to the Hb S allele. Probes that matched either the wild-type or Hb C alleles did not resolve all genotypes as effectively. The total time for amplification and genotyping of 32 samples was <1 h.

Probe melting temperature is dependent on external factors (salt concentration and pH) and intrinsic factors (concentration, duplex length, GC content, and nearest neighbor interactions) (11)(12)(13)(14). Mismatches between the oligonucleotide probe and DNA target cause a decrease in the T_m of the duplex (15)(16). By designing a probe specific to a mutation locus, genotyping by melting curve analysis in real time is possible. Each allele has a characteristic T_m, and multiple alleles can be identified by a single probe. Depending on the type of mismatch, varying degrees of destabilization occur. Mismatches that are relatively stable, e.g., G-T mismatches, have T_m shifts of 2–3 °C (7), whereas less stable C-A mismatches have 8–10 °C T_m shifts (6)(8). The mismatch position and the number of mismatches also affect T_m and can be used to enhance polymorphism discrimination (16).

Single-color hybridization probes can identify multiple mutations under a probe and detect unexpected polymorphisms. The cystic fibrosis F508del locus was genotyped recently by melting curve analysis with hybridization probes (17). A 10 °C T_m shift was observed between the wild-type and the F508del alleles. Unexpectedly, some samples showed aberrant 5 °C T_m shifts. This unique T_m was correlated to the F508C allele with a single T-C mismatch to the probe. Subtle changes in T_m can also indicate unexpected polymorphisms. A probe to the factor V locus revealed two additional mutations sites characterized by differences in T_m as small as 0.8 °C (18). Because a single probe can characterize multiple alleles, we designed a single hybridization probe to cover the adjacent single base changes in exon 1 of the hemoglobin gene that produce Hb C and Hb S. Additional alterations in the sequence under the probes, such as Hb Leiden (deletion of codon 6 and/or 7) or Hb Chesterfield (codon 28 CTGCGG), should also be detectable (9).

Multiple hybridization probes can also be used for multiplex genotyping of loci that are not in close proximity. Multiplexed hybridization probes of the same color have identified four different hemochromatosis alleles and a fifth unexpected variant (8). Two probe pairs each identified two expected alleles with characteristic T_ms. The T_m of the unexpected variant was within 1.5 °C of a mutant allele. Although the T_ms of the five alleles were discernible in this case, unexpected mutations can cause genotyping errors if allele T_ms overlap. Although the number of distinct T_ms that can be differentiated is limited, different colors can be used to increase the extent of multiplexing. For example, hybridization probes have been used to detect apolipoprotein E mutations at the 2/3 and 3/4 loci with LC Red 640 and LC Red 705 dyes, respectively (19).

Other methods have been developed to detect polymorphisms with oligonucleotide probes. TaqMan^TM probes (20)(21) and molecular beacons (22) can be used to correlate a single fluorescent probe to a specific allele. In these cases, the number of different detectable mutations is limited to the number of colors available. Although not performed in real time (21), the TaqMan system has been used to genotype three different point mutations simultaneously by using seven different colored probes (one for background correction). By multiplexing the probe T_ms and only two colors, we were similarly able to genotype three different point mutations. An additional advantage of using hybridization probes is that unexpected sequence alterations can be detected.

The ability to multiplex PCR analysis by color and T_m has many uses in addition to multiplex genotyping. For example, internal amplification controls often are needed for infectious disease and translocation testing to verify that amplifiable DNA or cDNA is present even if the target amplification is negative. Another common need is for multiplexing a competitor as an internal standard for PCR quantification.

Acknowledgments

This work was financially supported by an STTR grant from NIH (GM-58983). We thank Alex Chagovetz, Christine Litwin, and Elaine Lyon for insightful conversation, and Doug Searles for probe synthesis.

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