Molecular genetic assays

An introduction to single gene transcript quantification methods. A number of methods are now available for the detection of gene activity via messenger RNA (mRNA). In all protocols, the first step is to isolate total RNA. This can be probed directly or used for reverse transcriptase polymerase chain reaction (RT-PCR) 

Fluorescence-based quantitative PCR. Initially, a major obstacle to the use of PCR for gene expression quantification was that the final level of product that could be detected was derived from a limitation not related to the starting quantity of template. This means that parallel reactions with vastly different template inoculations resulted in near identical final product levels. To overcome this problem, techniques have been developed that take a snap shot of product levels during the PCR reaction. The most commonly used of these are based on fluorescence in situ monitoring of PCR progress using two main detection methods. 

SYBR Green dye, which fluoresces when bound inside the double helix of DNA. 

The fluorogenic 5 nuclease assay, which utilises the 5 nuclease activity inherent as a secondary function of Taq DNA polymerase to cleave a gene-specific probe, thereby releasing a fluorescent molecule for detection. 

For both detection systems, the inclusion of a series of calibration standards containing cloned copies of the target gene at known concentrations can be used to obtain the relationship between transcript frequency and the number of cycles required to obtain a specific threshold. This standard curve is then used to determine gene concentration in samples. 

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Molecular genetics assays can be designed to amplify both normal and mutant alleles, then to determine which allele is present in a postamplification phase of the analysis. For detection of single base changes, a generic amplification is conducted with primers that flank the site of the mutation. Following PCR, oligonucleotide probes for wild type or mutant are... 

Herzberg, M. (1984) Molecular genetic probe, assay technique, and a kit using this molecular genetic probe. European Patent Application, 0128018. 

Wegener, D., Hildmann, C. and Schwienhorst, A. (2003) Recent progress in the development of assays suited for histone deacetylase inhibitor screening. Molecular Genetics and Metabolism,... 

One of the primary responsibilities of the director of a molecular genetics testing center is to make certain that appropriate controls are devised for and run with each diagnostic assay. An elaborate system of checks exists in most genotyping laboratories in order to make certain that each released result meets a written QC standard. In the absence of QA/QC, patients, physicians, and researchers cannot rely upon the genotypes being produced. 

The workload is increasing in both volume and scope in all branches of laboratory medicine. In mainstream clinical chemistry, the past 25 years have seen astonishing developments in assay technology and instrumentation assays that were performed manually with a few dozen assays a day can now be accomplished automatically by the thousands. In biochemical genetics (molecular genetics aside), automation has had a less dramatic impact. 

Feigenbaum A, Moore R, Clarke J, Hewson S, Chitayat D, Ray PN, Stockley TL. Canavan disease carrier frequency determination in the Ashkenazi Jewish population and development of a novel molecular diagnostic assay. Am J Med Genet 2004 124A 142-147. 

Miller JH (1972), Assay of p-galactosidase, In Miller JH (Ed.), Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 352-355. 

Before molecular tools have been widely available, strains had to be isolated and cultivated to determine toxin production. More recent studies employing either molecular genetics tools such as mcy-specific PCR, mass spectrometry, or immunosorbent assays on single filaments or colonies indicate that a coexistence of multiple clones, a number of which produce MCs, is typical for most water bodies infested with cyanobacteria rather than an exception. ... 

Amperometry at single PC 12 cells has also been used in conjunction with a genetic cell transfection protocol to examine the effects of toxin expression on basal and evoked exocytosis. PC 12 cells have been transfected with the specific endoprotease Botulinum neurotoxin Cl light chain (BoNT/Cl), which cleaves the proteins syntaxin and SNAP-25 [5], The molecular dissection of the mechanisms underlying exocytosis has been motivated by the SNARE hypothesis, which postulates that exocytosis requires the assembly of the plasma membrane proteins syntaxin 1, SNAP-25, and the vesicle associated membrane protein (VAMP) into a complex [5], This SNARE complex then acts as a receptor for cytosolic components of the proposed fusion machinery. Direct evidence for the role of the SNARE proteins in neurotransmission comes from molecular genetic studies in which syntaxin and VAMP have been shown to be required for neurotransmission in Drosophila [47 9] and Caenorhabditis elegans [50,51]. To assess the effects of the disruption of SNARE proteins on exocytosis in PC 12 cells, amperometry has been used in conjunction with a genetic cell transfection assay to establish a... 

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