Amplification of DNA

R. P. Lcite, G. U. Miinsavage, U. Bonas, and R. E. Stall, Detection and identification of phytopathogenic Xanthomonas species by amplification of DNA-.sequences related to the HRP genes of Xanthomonas-campestris pv. vesicatoria. Appl. Environ. Microbiol. 60 1077 (1994),... 

Molecular methods used to uncover mutations are subject to several variables. The anticoagulants used for blood collection can affect digestion with restriction enzymes and amplification reactions. The type of detergent used in cell lysis can affect amplification of DNA by inhibiting the DNA-amplifying enzyme such as the taq polymerase used in the polymerase chain reaction (116). The control of contamination is crucial in ensuring the quality of results obtained by molecular analysis (117). 

Saiki, R. K., etal. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239,487-491. 

Mullis, K.B., Faloona, F., Scharf, S., Saiki, R., Horn, G. and Erlich, H. (1986) Specific enzymatic amplification of DNA in vitro the polymerase chain reaction. Cold Spring Harbor Symposium of Quantitative Biology 51, 263—273. 

The process known as SPREAD (Surface Promoted Replication and Exponential Amplification of DNA Analogues) attempts to reach the target, striven for by many researchers, of an exponential proliferation of biomolecules in model systems. As already mentioned, product inhibition (e.g., by dimerisation of the new matrices to give C2) only allowed parabolic growth. In the SPREAD process, both solid phase chemistry and feeding have a positive effect on the synthesis. Thus, no separation processes are required, as excess reagents can be removed just by washing. The synthetic process consists of four steps ... 

SPREAD surface promoted replication and exponential amplification of DNA analogues... 

Faulkner SW, Leigh DA. Universal amplification of DNA isolated from small regions of paraffin-embedded, formalin-fixed tissue. BioTechniques 1998 24 47-50. 

PCR is a technique for in vitro amplification of DNA sequences that involves repeated cycles of denaturation, oligonucleotide annealing, and DNA polymerase extension [29], The amplified products following PCR cycles contain double-stranded DNA fragments of discrete length. These DNAs are copies of the template DNA that are bounded at the 5 -terminus by the oligonucleotide primer for the sequence extension with a heat-resistant DNA polymerase. In quantitative assays of PCR products, therefore, nonspecific products interfere with the assay. 

Arbeli Z, Fuentes CL (2007) Improved purification and PCR amplification of DNA from environmental samples. FEMS Microbiol Lett 272 269-275... 

Figure 13.16 The polymerase chain reaction for the amplification of DNA sequences. DNA is heated to separate the two strands. A primer is attached to the 5 end of each strand and extended using DNA polymerase 1. The two new strands are separated as before and the cycle repeated up to 30 times.

RNA amplification by PCR has been facilitated by the use of a single heat-stable enzyme. Thus, DNA polymerase from Thermus thermophilus, which has enhanced reverse transcriptase (rT) activity in presence of manganese, can be used with one buffer system. The high temperature used for rT (70°C) to produce a complementary DNA copy from RNA, and the subsequent amplification of DNA at 60°C, increases efficiency by destabilizing secondary structures in the RNA template. This procedure has been used for the amplification of hepatitis C viral RNA (Yl). 

The PCR phase of such a study is very similar to PCR amplification of DNA in a reaction tube placed in a thermocycler. Here, however, the specimen is affixed to a slide covered with the reactants (buffer, DNTPs, primers, and a thermostabile DNA polymerase) and placed on the heating block of a traditional or modified (for slides) thermocycler programmed to provide the optimum temperatures for denaturation, primer annealing, and extension. After amplification for 20-30 cycles, the specimen is processed for ISH. 

Definition of a plasmid Function in amplification of DNA Prokaryotic organisms normally contain small, circular, extrachromosomal DNA molecules called plasmids that can serve as vectors. They can be readily isolated from the bacterium, annealed with the DNA of interest, and reintroduced into the bacterium which will replicate, thus making multiple copies of the hybrid plasmid. 

Amplification of DNA of chromosomes. During formation of oocytes parts of the DNA are "amplified" by repeated replication. This provides a way for the ovum to accumulate ribosomal RNA and various proteins in large amounts. Similarly, genes for two abundant proteins of the egg shell or chorion of insects are amplified. Bidirectional replication initiated at discrete positions yields an "onion skin" structure containing many copies of an 90-kb sequence containing the two genes. The polyploidy observed in some highly specialized cells such as the Purkinje cells... 

T. Lumley-Woodyear, C.N. Campbell, E. Freeman, A. Freeman, G. Georg-iou and A. Heller, Rapid amperometric verification of PCR amplification of DNA, Anal. Chem., 71 (1999) 535-538. 

Figure 6.6. Amplification of DNA by PCR. Target DNA sequence from a complex genome can be amplified by heat denaturation, providing appropriate conditions for the enzyme (Taq DNA polymerase) that allow it to cause exponential amplification of a particular DNA segment. Among components besides the enzyme that are essential for amplification process are oligonucleotide primers in opposite orientation to each other, shown by dotted arrows, deoxynucleotide triphosphates (dNTPs), Mg2+, and buffer. A 30-cycle amplification leads to a many-million-fold amplification of the discrete DNA segment, flanked by oligonucleotide primer sequences. (Reproduced from Short Protocols in Molecular Biology, 4th ed., F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds., Wiley, New York, 1999, p. 15-1.)...

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]. 

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