ABSTRACT
The Invader assay (Third Wave Technologies) is a homogeneous, isothermal DNA probe-based method for sensitive detection of nucleic acid sequences. Invader reactions are performed directly on genomic DNA or total RNA targets; however, polymerase chain reaction- or reverse transcriptase polymerase chain re action-amplified products can also be used. Detection is achieved through target-specific signal amplification instead of target amplification. The assay is a highly accurate and specific detection method for both qualitative and quantitative analysis of single-nucleotide changes, insertions or deletions, gene copy number, infectious agents, and gene expression.
Subject(s)
DNA/analysis , Gene Dosage , Molecular Probe Techniques , Polymorphism, Single Nucleotide , Fluorescence Resonance Energy Transfer/methods , Fluorescent Dyes , Genotype , Molecular ProbesABSTRACT
With the use of synthetic biology, we reduced the Escherichia coli K-12 genome by making planned, precise deletions. The multiple-deletion series (MDS) strains, with genome reductions up to 15%, were designed by identifying nonessential genes and sequences for elimination, including recombinogenic or mobile DNA and cryptic virulence genes, while preserving good growth profiles and protein production. Genome reduction also led to unanticipated beneficial properties: high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Eradication of stress-induced transposition evidently stabilized the MDS genomes and provided some of the new properties.
Subject(s)
Escherichia coli K12/genetics , Gene Deletion , Genome, Bacterial , DNA Transposable Elements , DNA, Bacterial , Genetic Engineering , Mutagenesis , Plasmids/genetics , Species SpecificityABSTRACT
Concomitant advances made by the Human Genome Project and in the development of nucleic acid screening technologies are driving the expansion of pharmacogenomic research and molecular diagnostics. However, most current technologies are restrictive due to their complexity and/or cost, limiting the potential of personalized medicine. The invader assay, which can be used for genotyping as well as for gene expression monitoring without the need for intervening target amplification steps, presents an immediate solution that is accurate, simple to use, scaleable and cost-effective.
Subject(s)
DNA/analysis , Genetic Techniques , Molecular Diagnostic Techniques , RNA/analysis , Alleles , Automation , DNA/metabolism , DNA Mutational Analysis , Genotype , Humans , Models, Genetic , Polymorphism, Genetic , RNA, Messenger/metabolism , Sensitivity and SpecificityABSTRACT
The cytochrome p450 (CYP) superfamily comprises enzymes that play an essential role in the transformation of medically relevant compounds. Accurate genotyping of polymorphisms in members of this family is drawing increasing interest because certain allelic variants may result in either loss of efficacy or toxic accumulation of therapeutic agents. Debrisoquine 4-hydroxylase, or CYP2D6, is among the most widely studied of the CYPs. The complexity of the CYP2D6 genomic region, including pseudogenes, gene deletions, and gene duplications, has offered numerous challenges to developing a genotyping strategy. We describe a comprehensive CYP2D6 genotyping strategy that employs both a PCR/Invader genotyping assay system and an Invader genomic copy number assay The Invader system is a homogeneous, isothermal, highly specific, and robust signal amplification system. Resultsfrom II CYP2D6 assays in an alle frequency study compare well to published allele frequency values for Caucasians. Further, Invader assays provided unambiguous genotyping determinations for 100% of the 171 samples that yielded a visible PCR product on an agarose gel. A copy number assay yielded only one equivocal result in 205 samples. We identified 17 single-copy individuals and 17 three-copy (or more) individuals.
