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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 for
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