( Three new kits tar...)Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations

Three new kits target specific mutations using molecular beacon technology

Xiuyuan Hu Beti Belachew Lingyu Chen Haoqiang Huang Jason Zhang
Stratagene

Because of their unique hairpin conformation, molecular beacons can distinguish human DNA specimens that differ by a few nucleotides or only a single nucleotide. We developed three single-tube assay kits, based on molecular beacon technology, to quickly detect three common human gene mutations: One is a three-base-pair deletion in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the other two are single nucleotide substitutions in the coagulation factor II (prothrombin) gene and the HFE gene. Each kit includes two allele-specific molecular beacons, three genotype-specific DNA controls, target-specific PCR primers, and an optimized PCR buffer. Test results show that these kits possess adequate target specificity and good assay throughput.

Recent advances in the human genome project and in other areas of genetic research have revealed the value of single nucleotide polymorphisms (SNPs) in medical research and clinical diagnostics. The demand is growing for high-throughput screening methodologies that are low in cost yet specific enough to distinguish nucleic acid sequences differing by a single nucleotide. Currently used mutation detection techniques are either labor intensive and/or expensive (e.g., DNA sequencing) or do not detect all mutations (e.g., allele specific amplification and single-strand confirmation assay). However, molecular beacon technology overcomes these limitations.^1,2

Molecular beacons are single-stranded oligonucleotides that possess a stem-and-loop hairpin structure. The loop portion of the molecule is a probe sequence, which is complementary to a target sequence, and the stem is formed by short complementary sequences located at the opposite ends of the molecule. The molecule is labeled with a fluorophore at one end and a quencher at the other end. When the unhybridized probe is in solution, it adopts a hairpin structure that brings the fluorophore and quencher sufficiently close to each other to allow efficient quenching of the fluorophore. When the molecular beacon is bound to its complementary target, the fluorophore and quencher are far enough apart so that the fluorophore cannot be quenched, and the molecular beacon fluoresces (Figure 1). The hairpin shape of the probe causes mismatched probe/target hybrids to easily dissociate at a significantly lower temperature than perfectly matched hybrids.²

Fig.1

The thermal instability of mismatched hybrids increases the specificity of molecular beacons, enabling them to distinguish targets that differ by only a single nucleotide. When conjugated to different fluorophores, two allele-specific molecular beacons can simultaneously discriminate the three possible genotype representations (two homozygotes and a heterozygote) of two allelic variants in a single reaction tube (Figure 1).

Convenient Single-Tube Format

Each assay kit includes two allele-specific molecular beacons with an exact sequence match to each of the two target sequence variants, genotype DNA controls (wild type/mutant homozygotes and heterozygote), target-specific PCR primers, and an optimized PCR buffer. The wild-type allele-specific molecular beacon is labeled with the fluorophore tetrachloro 6-carboxyfluorescein (TET), and the mutant allele-specific molecular beacon is labeled with the fluorophore 6-carboxyfluorescein (FAM). DABCYL is used as the quencher on both molecular beacons.

Fig.2

The assay is performed in a single PCR tube that contains a DNA template, both allele-specific molecular beacons and other PCR reagents. We carried out the amplification in the ABI Prism 7700 sequence detector. The genotype of a DNA sample is determined by measuring the endpoint fluorescence value (endpoint analysis, Figure 2) and/or the threshold cycle (Ct) value (real-time analysis) of the sample. Monitoring PCR reactions in real time produces a fluorescent signal during each cycle. Molecular beacon fluorescence is reported during each annealing step when the molecular beacon is bound to its complementary target (Figure 3 and Figure 4).

Results are displayed as an amplification plot that reflects the change in fluorescence during cycling. The threshold cycle is the cycle at which the fluorescent signal is first detectable above background. For the three assays described in the article, a Ct value of 20 to 32 indicates the presence of a specific allele and a Ct value of 40 indicates the absence of a specific allele. The maximum Ct value is 40 as PCR proceeds to 40 cycles.

The Specific Kits

The CFTR (DF508) Mx4000 molecular beacon allelic discrimination kit^ff, is designed to rapidly detect/screen the most common mutation that causes cystic fibrosis (CF). This mutation, a three-base-pair deletion, within exon 10 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, accounts for approximately 70% of all mutant CF alleles.^3,4 The Factor II (G20210A) Mx4000 molecular beacon allelic discrimination kit^ff, is used to detect a common genetic variant in the coagulation factor II (prothrombin) gene. The mutation, a G to A substitution at nucleotide position 20210, is associated with elevated plasma prothrombin levels and an increased risk of deep vein thrombosis.^5,6 The mutation is also being evaluated clinically as a risk factor for cardiovascular disease.⁷

The HFE (C282Y) Mx4000 molecular beacon allelic discrimination kit^ff, is used to detect a G to A substitution at the nucleotide position 845 of the HFE gene.⁸ This mutation, which changes cysteine to tyrosine at amino acid codon 282, is found in homozygous form in more than 80% of Caucasian patients suffering from hereditary haemochromatosis. One of the most common human genetic diseases, it characterized by an iron overload that eventually results in tissue damage and death to the patient. The estimated frequencies of the disease varies from 1 in 200 to 1 in 400 in individuals of Northern European descent.⁹

Assay Validation

Fig.4

The assay specificity of the three kits was determined by real-time PCR analysis using the ABI Prism 7700 sequence detector. First, validated genotype-specific human genomic DNA was used as PCR template, and the expected results were obtained by molecular beacon analysis (assay details in kits manual). Examples of the results for CFTR (DF508) allelic discrimination are shown in Figure 2 and Figure 3. An example of the results for factor II (G20210A) allelic discrimination is shown in Figure 4. Second, 50 human genomic DNA, previously genotype-characterized by PCR/RFLP analysis for the factor II and the HFE mutations, were tested, and results of the molecular beacon analysis were in complete agreement with those from the PCR/RFLP analysis. Third, 154 genotype-unknown human genomic DNA samples were genotyped by molecular beacon analysis for the three mutations, and results were confirmed for selected samples by DNA sequencing.

Conclusions

Use Stratagenes single-tube allelic discrimination assays to realize several advantages over existing mutation detection techniques: The hairpin-shaped molecular beacon probes are more specific in distinguishing single base-pair mismatches than linear probes.² Because the test is performed in a closed tube and no post-PCR manipulation of samples is required, the test saves time and effort and significantly reduces the risk of PCR product carry-over contamination. Being able to use two allele-specific molecular beacons in the same PCR solution enables three possible genotype representations (two homozygotes and a heterozygote) of two allelic variants in target DNA to be determined at the same time. It also definitively discriminates a true negative result from a false negative result due to PCR failure. Finally, the technology can be adapted for high-throughput assays. Currently, with the 96-well PCR plate, it takes about 4 hours to completely screen 96 DNA samples; further enhancement of the assay throughput can be achieved by using a higher density plate format (e.g., 384-well format).

Acknowledgments

The authors thank the following: Dr. P.N. Ray (Molecular Diagnostic Laboratory, the Hospital for Sick Children, Toronto, Canada) for providing the CF genotype-specific human genomic DNA; Dr. J.W. Longshore (Molecular Diagnostic Laboratory, Greenwood Genetic Center, Greenwood, SC) and Dr. V.M. Pratt (LabCorp, Research Triangle Park, NC) for providing the factor II genotype-specific human genomic DNA; Dr. J.W. Longshore (Molecular Diagnostic Laboratory, Greenwood Genetic Center, Greenwood, SC) and Dr. C. Mura (Laboratoire de Genetique Moleculaire, CHU-UBO, France) for providing the HFE genotype-specific human genomic DNA; and Dr. Cindy WalkerPeach, Martha Frantz, Jeff Strauss, Peter Pingerelli, Dwight Dubois, David Boe, Connie Hansen, Becky Mullinax, and Joe Sorge of Stratagene for discussions, suggestions, and reagents.

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