Polymerase based methods

Two principal types of nucleic acid-based methods, nucleic acid hybridization and polymerase chain reaction (PCR), are commonly used for the rapid identification of bacteria. A few other nucleic acid-based methods will also be mentioned. 

Antibody-based detection methods include immuno-cytochemistry, which gives qualitative data but has very good spatial resolution. Radioimmunoassays provide a quantitative measure of release or content. One of the major limitations of all antibody-based methods is the potential for cross-reactivity among the many peptides. For example, some of the most sensitive gastrin antisera also detect CCK, since the peptides share a common COOH-terminal tetrapeptide sequence. Methods for detection of the mRNAs encoding neuropeptides include Northern blots, which provide quantitative data and information on splice variants, but lack fine anatomical resolution. The more commonly used polymerase chain reaction, which can be quantitative but often is used in a more qualitative manner, provides great sensitivity. Alternatively, in situ hybridization preserves anatomical relationships and can be used to obtain both qualitative and quantitative data. 

B3. Begovich, A. B., and Erlich, H. A., HLA typing for bone marrow transplantation. New polymerase chain reaction-based methods. JAMA, J. Am. Med. Assoc. 586-591 (1995). 

Venous blood (8 mL) was collected into 15-mL tubes containing 50 mmol/L disodium EDTA, and genomic DNA was extracted by the standard (phenol/chloroform) method. The genotype frequencies for GSTPl were determined by polymerase chain reaction (PCR)/RFLP-based methods using DNA extracted from peripheral lymphocytes. 

Another class of readout measures RNA expression levels, with the three most common methods being chip-based hybridization/fluorescence techniques, realtime polymerase chain reaction (RT-PCR) and quantitative nuclease protection assays (QNPA) [48, 49]. Chip-based methods are widely used for whole-genome scans (discussed in more detail below), but have a disadvantage that they are relatively expensive and so are not really high throughput. The quantitative reproducibility and dynamic range of these chip-based methods are also lower than for the other RNA readout techniques. RT-PCR is a more quantitative technique for measuring transcript levels, and is typically run for up to 40 transcripts at a time. QNPA is another... 

Finally, several PNA based methods for use in genetic diagnostics have been developed. Of particular interest are methods for fluorescence in situ hybridization (FISH), modulation of polymerase chain reaction (PCR) analyses, and detection of genetic material by sensors and mass spectrometry. 8 ... 

Different protein-based methods have been reviewed for species identification in milk and dairy products, and for characterization of cheese maturity, such as electrophoretic, chromatographic, and immunological techniques (11, 12). In addition to new developments in these techniques, the interdisciplinary and dynamic nature of milk product analysis is being enhanced by the application of disciplines already used to analyze other foodstuffs. Among them, capillary electrophoresis (CE), polymerase chain reaction, and isotope ratio mass spectrometry are just gaining popularity for solving dairy authenticity problems (13-15). 

In recent decades a number of polymerase chain reaction (PCR)-based methods for fish species identification by DNA analysis have been developed to support food control authorities to achieve this goal. In the following sections these techniques are described... 

De Roo G, Ren Q, Witholt B, Kessler B (2000) Development of an improved in vitro activity assay for medium chain length PHA polymerase based on coenzyme A release measurements. J Microbiol Methods 41 1—8... 

In addition to PCR, there are many other technologies to amplify nucleic acids. For example, ligation-based amplification or ligase chain reaction uses sequence-directed oligonucleotide primers and thermostable DNA ligase to assay point mutations, deletions, or insertions in DNA. Strand-displacement amplification uses the inherent strand-displacement activity of DNA polymerases to conduct DNA amplification at a constant temperature. Transcription-based methods such as nucleic acid sequence-based amplification (NASBA) involve in vitro RNA transcription. NASBA and most other transcription-based... 

Polymerase chain reaction (PCR)-based methods PCR (specific primers) PCR-RFLP of the 5.8s ITS rDNA region Species Species... 

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