DNA amplification method

Palmer HM, MaUinson H, Wood RL, Herring AJ, Evaluation of the specificities of five DNA amplification methods for the detection of Neisseria gonor-rhoeae. J Clin Microbiol 2003 41 835-7. 

There are three types of sample contamination that can result in false positives with DNA amplification methods. Two types are familiar to clinical chemists. For... 

Methods for meat species identification based on DNA analysis benefit from the heat stability of the DNA molecule and its high specificity. Originally, DNA methods consisted of immobilization of partially purified and denatured DNA, extracted from the meat product sample, on a nylon membrane, followed by hybridization of a species-specific segment of labeled (colorimetric, fluorescent, or chemiluminescent) DNA with any complementary sequences of DNA present on the membrane. More recently, a DNA amplification method - the polymerase chain reaction - has been used, but this is a relatively expensive and technically demanding technique. 

EL-MATBOULI M and SOLIMAN H (2005), Rapid diagnosis of Tetracapsuloides bryo-salmonae, the causative agent of proliferative kidney disease (PKD) in salmonid fish by a novel DNA amplification method, loop-mediated isothermal amplification (LAMP), Parasitol Res, 96(5), 277-284. 

Sato Y, Sugie R, Tsuchiya B, et al. Comparison of the DNA extraction methods for polymerase chain reaction amplification from formalin-fixed and paraffin-embedded tissues. Diagn. Mol. Pathol. 2001 10 265-271. 

FIGURE 14.2 The bio-bar-code assay method, (a) Probe design and preparation, (b) PSA detection and bar-code DNA amplification and identification (reproduced from [17] with permission) (see Plate 14 for color version). 

Methods of this nature are adequate for screening sets of hybridomas but not for selection from much larger libraries of antibodies. So, most recently, selection methods employing suicide substrates (Section 7) (Janda etal., 1997) or DNA amplification methodology (Fenniri et al., 1995) have been brought into the repertoire of techniques for the direct identification of antibodies that can turn over their substrate. However, the tedious screening of hybridomas remains the mainstay of abzyme identification. 

In 1989, a very practical mutagenesis method was described by Leung et al. 39), which was later improved by Cadwell and Joyce 40). Error-prone polymerase chain reaction (epPCR), as it is called, is based on the classical DNA amplification... 

Fig. 3. The use of microdissected material increased the sensitivity of the SNP array to LOH in tumor tissues. Regions with LOH are highlighted in black color. B, DNA extracted from bulk tumor tissue without microdissection M, DNA was extracted from microdissected tumor cells and amplified by the multiple displacement amplification method (52).

Real-time RNA analysis is also possible by these methods, once the RNA species have been converted to cDNA using reverse transcription. Thermostable reverse transcriptase enzymes are now available commercially, allowing one-step reverse transcription and DNA amplification (184-187). 5... 

Methods for Amplification of Select Segments of DNA Amplification by the Polymerase Chain Reaction DNA Cloning... 

Wood et al. (1991) have used the Southern hybridization method for detecting DNA amplification and a possible structural rearrangement of the HER-2/nen oncogene in 1 of 12 bladder tumors. Amplification of this oncogene in the tumor was sixfold that of oncogene found in placental DNA. Approximately 36% of the tumors studied overexpressed HER-2 mRNA, which was 3- to 38-fold that of normal urothelium. HER-2 overexpression occurred in superficial and invasive tumors. Deoxyribonucleic acid amplification occurs infrequently in bladder carcinoma, in contrast to its occurrence in some other carcinomas. Immunohistochemical analysis has shown that pi85 HER-2 polyclonal antibody is specific for HER-2 protein overexpression in bladder carcinoma. This study was carried out prior to the use of Herceptin. 

However, real-time detection requires access to a special real-time PCR cycler, which is able to detect the increase/decrease of added fluorescence labels during DNA amplification. Although these machines are more and more common for quantitative DNA analysis, their availability in clinical laboratories is still limited. Therefore, the following subsections also include a detailed overview of the classical approaches to quantitative (I)PCR amplificate, analysis which exchanges less demanding PCR equipment for additional hands-on time. The sensitivity of real-time or end-point IPCR detection is quite similar. A comparison of the influence of different endpoint detection methods to the overall sensitivity of IPCR is given in Fig. 5. 

Starting with the first IPCR study, gel electrophoresis retains its potential as a fast and easy method for end-point determination of DNA amplificate for IPCR assays [10, 24, 25, 29, 31, 35, 36, 38, 39, 64], Readout is performed by intercalation fluorescence markers (e.g., ethidium bromide) and photometric/densitometric quantification of band signal intensities. The direct addition of a double-strand specific intercalation marker to the PCR amplificate and subsequent measurement of fluorescence in microwells proved to be of insufficient sensitivity for the quantification of IPCR amplificate [37]. Alternative approaches, such as radioactive labeling during PCR and subsequent imaging [33], were carried out but are not well suited for routine clinical application because of additional methodological requirements. An advantage of gel electrophoresis is the possibility of simultaneous amplificate detection for multiplex IPCR [41] and the ability to detect nonspecific amplification products. 

A major breakthrough in the development of quantitative PCR was the invention of real-time detection methods for DNA amplification product during PCR [63], In this technique, a fluorescence-generating probe (e.g., a Taq-man probe, see Fig. 7A) is added to the PCR mix. During amplification of the DNA template, the probe is modified/degraded, so that an initially quenched fluorescence increases parallel to the increased amount of amplified DNA. As a less specific alternative, an intercalation marker could be added to the PCR mix, which incorporates in the double-stranded PCR product and thereby increases fluorescence during PCR. 

The polymerase chain reaction is the prevalent method for DNA amplification. Much effort has been made to integrate PCR chambers on microchips to carry out amplifications of DNA molecules prior to their analysis. For instance, PCR was first achieved on a Si-based reaction chamber (25 or 50 pL) integrated with a polysilicon thin-film (2500-A-thick) heater for the amplification of the GAG gene sequence (142 bp) of HIV (cloned in bacteriophage M13) [997]. 

---