Allelic discrimination kits with excellent specificity, sensitivity, and assay throughput
Xiuyuan Hu Lingyu Chen
Because of their unique hairpin conformation, molecular beacon probes can recognize single base differences, making them ideal for mutation detection and allelic discrimination. Based on the molecular beacon technology, we developed a series of Mx4000 molecular beacon allelic discrimination kits,ff for the detection of single or multiple nucleotide changes in human genes. The five kits described in this article allow four single base substitutions to be detected in the human CCR2 (64I), Factor V (Leiden), SDF-1(3 A), and MTHFR (C677T) genes, as well as of a 32-base-pair deletion in the human CCR5 (D32) gene. Each kit includes two allele-specific molecular beacons in addition to genotype-specific DNA controls, PCR primers, and optimized target amplification buffer. The two molecular beacons are labeled with different fluorophores that emit fluorescent light at distinct optical wavelengths. As a result, three possible allelic combinations of two sequence variants (Allele 1/Allele 1, Allele 2/Allele 2, Allele 1/Allele 2) can be distinguished simultaneously in a DNA sample. The technique saves time and reduces the risk of contamination as the test is performed in closed tubes and requires no post-PCR manipulation of samples.
Single nucleotide sequence changes (substitution, deletion, or insertion) are the largest source of human DNA diversity, with an estimated frequency of 1 in 1,000 base pairs. Many of the sequence changes have been identified as the cause of monogenic disorders or are associated with a genetic predisposition to multifactorial diseases including cancer and cardiovascular and infectious diseases. The sequence changes also constitute the genetic basis for many nondisease traits, such as obesity and a persons response to drugs. Moreover, single nucleotide polymorphisms (SNPs) are valuable genetic markers to use for gene discovery, population studies, and individual identification. The demand is growing in the clinical research field for high-throughput screening methodologies that are sensitive enough to distinguish nucleic acid sequences differing by a few or one nucleotide. The recently described molecular beacon technology has demonstrated the potential to meet such a challenge.1,2
Molecular beacons are hairpin-shaped oligonucleotide probes that fluoresce when they hybridize to their target (Figure 1). The hairpin shape of the molecular beacon causes mismatched probe/target hybrids to easily dissociate at a significantly lower temperature than exact complementary hybrids.2 This thermal instability of mismatched hybrids increases the specificity of molecular beacons, thus enabling them to distinguish targets that differ by a few or only a single nucleotide. When conjugated with different fluorophores, molecular beacons can be used to differentiate different target sequences in the same solution.
Stratagenes Mx4000 molecular beacon allelic discrimination kits include the following components: a mixture of two allele-specific molecular beacons with an exact sequence match to each of the two target sequence variants, three genotype-specific DNA controls corresponding to the homozygotes and the heterozygote of the two sequence variants, target-specific PCR primers, and an optimized PCR buffer.
The two allele-specific molecular beacons are labeled with different fluorophores. The molecular beacon specific to the wild-type al lele is labeled with the fluorophore tetrachlorofluorescein (TET), and the molecular beacon specific to the mutant allele is labeled with the fluorophore 6-carboxyfluorescein (FAM). DABCYL is used as the quencher on both molecular beacons. By using two different molecular beacons in each PCR reaction, three possible allelic combinations (genotypes) of two sequence variants can be distinguished simultaneously by the type of fluorescence detected: TET fluorescence indicates homozygosity for the wild-type allele, FAM fluorescence indicates homozygosity for the mutant allele, and both TET and FAM fluorescence together indicates heterozygosity (Figure 2).
Screening is performed with PCR mixtures that consist of a DNA template, both allele-specific molecular beacons, PCR primers, dNTPs, Taq2000 DNA polymerase, and the optimized PCR buffer. Amplification is carried out in a spectrofluorometric thermal cycler. The alleles in a DNA sample are determined by comparing the endpoint fluorescence value (Figure 3) and/or the threshold cycle (Ct) value (Figure 4 and Figure 5) of the DNA sample to the endpoint fluorescence values and/or Ct values of the three genotype-specific DNA controls.
Three kits (CCR2-64I allelic discrimination kit, CCR5-D32 allelic discrimination kit, and SDF-1-3A allelic discrimination kit) are used to detect three common sequence variants that occur in the human CCR2, CCR5, and SDF-1 genes. These variant s have been correlated with the delayed or accelerated onset of AIDS in HIV-1 infected individuals (Table 1). The Factor V (Leiden) allelic discrimination kit is used to detect a common point mutation (Factor V Leiden) in the human Factor V gene. Carriers of this mutation are at significantly increased risk of venous thrombosis (Table 1). The MTHFR (C677T) allelic discrimination kit is used to detect a common point mutation in the human methylenetetrahydrofolate reductase gene. This mutation has been shown to be an important genetic risk factor for vascular disease and coronary heart disease (Table 1).
Consequence of Mutation
Chemokine receptor and a co-receptor for certain M-, T-, and dual-tropic strains of HIV
G to A substitution that results in Val to Ile change at position 64 (CCR2-64I)3
African Americans (~15%)3
Delays AIDS onset in HIV-1 infected individuals3,4
Chemokine rec eptor and major co-receptor for T-tropic HIV-1
A 32-bp deletion that results in a truncated protein5
Europeans (2 15%)6
Delays AIDS onset in HIV-1 infected individuals5, 7, 8
Inactivation of activated protein C (APC), an anticoagulant enzyme
G to A substitution that results in Arg to Gln change at position 506 (Factor V Leiden)11
Caucasians (2 - 5%)11, 12
Mutant carriers have a 7-fold higher risk for deep vein thrombosis11
A key enzyme in homocysteine metabolism
C to T substitution at nucleotide position 677 that results in an Ala to Val change13
Individuals of mutant homozygote have an elevated plasma homocysteine level, a major risk factor for cardiovascular diseases14,15
ligand of CXCR4, the major co-receptor for T-tropic HIV-1
G to A substitution in the 3 untranslated region (SDF-1-3 A) 9
African Americans (~5%)9
Mutant homozygote (3 A/3 A) is associated with delayed9 or accelerated10 AIDS progression
Molecular beacon technology has the following advantages over many mutation detection techniques. First, the hairpin-shaped probes are more specific than linear probes, such as the TaqMan probe,16,17 in distinguishing single base pair mismatches. Second, because the test is performed in closed tubes and no post-PCR manipulation of samples is required, the risk of PCR product carry-over contamination is greatly reduced. In addition, the time and effort involved in carrying out the test is significantly reduced. Third, the capability of using two allele-specific molecular beacons in the same PCR solution enables the simultaneous determination of three possible allelic combinations of two sequence variants in target DNA. It also definitively discriminates a true negative result from a false negative result due to PCR failure. Thus, the hairpin-shaped fluorescent probes are particularly useful for high-throughput mutation detection and genotyping assays.
The authors thank the following: Cindy WalkerPeach, Beti Belachew, Jeff Strauss, Peter Pingerelli, and Dwight Dubois of BioCrest (Austin, Texas) for providing DNA samples, comparing genotyping results, and offering suggestions; Michelle Cayouette, Ali Mousavi, Jane Moores, Haoqiang Huang, Jason Zhang, Jack Anderson, Lisa Grismer, Ronda Allen, SanDEe Soares, Connie Hansen, Becky Mullinax, and Joe Sorge of Stratagene; and Sanjay Tyagi of the Public Health Research Institute in New York for discussions, suggestions, and reagents.
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