Polymerase chain reaction inhibitors

List of Abbreviations PCR, polymerase chain reaction RT-PCR, reverse transcription polymerase chain reaction DNA, deoxyribonucleic acid RNA, ribonucleic acid RNase, ribonuclease mRNA, messenger RNA GABAa, y-aminobutyric acid type A cRNA, copy RNA dNTPs, deoxy nucleoside triphosphates MMLV, Mouse Moloney murine leukemia vims RT, reverse transcriptase bp, base pair Tm, melting temperature DEPC, diethylpyrocarbonate OD, optical density mL, milliliter SA-PMPs, streptavidin paramagnetic particles dT, deoxy thymidine DTT, dithiothreitol DNase, deoxyribonuclease RNasin, ribonuclease inhibitor UV, ultraviolet TBE, Tris-borate, 1 mM EDTA EDTA, ethylenediaminetetraacetic acid Buffer RET, guanidium thiocyanate lysis buffer PBS, phosphate buffered saline NT2, Ntera 2 neural progenitor cells... 

Nuovo, G. J., MacConnell, P. B, Simsir, A., Valea, F, and French, D. L. (1995) Correlation of the in situ detection of polymerase chain reaction-amplified metalloproteinase complementary DNAs and their inhibitors with prognosis m cervical carcinoma. Cancer Res. 55,267—275. 

The authors describe an ultrasensitive method that measures RT activity. The assay adopts polymerase chain reaction (PCR) amplification for detecting the cDNA product of the reaction, and therefore was named Amp-RT (3,4). Amp-RT measures the ability of a sample to produce a DNA copy of a known heteropolymeric RNA template by extending a complementary DNA oligoprimer. The RNA template used in Amp-RT is a sequence from the genome of the encephalomyocarditis virus. This chapter describes in detail the Amp-RT method and its use as (1) a qualitative assay for the generic detection of retroviruses, (2) a quantitative method to measure virus loads of the human immunodeficiency virus type 1 (HIV-1), and (3), a screening method for susceptibility of HIV-1 to RT inhibitors. 

All ingredients are present in the reaction mixture, which is added to a lipid film, and liposomes are prepared containing all macromolecules (enzymes and DNA or RNA templates) as well as all substrate molecules (nucleotides, for example). Consequently, this procedure has to be performed very quickly otherwise, the enzymatic reaction would mainly occur outside the liposomes and a distinction between product molecules synthesized inside from those produced outside and entrapped later would be difficult to draw. After the formation of liposomes, the enzymes outside the liposomes have to be inhibited by potent inhibitors (inhibitors that do their job even in the presence of substantial amounts of phospholipids) or the liposomal dispersion has to be treated by digestive enzymes. This strategy has basically been applied in the case of the RNA replication by QP replicase inside oleic acid/oleate liposomes" and in the case of the polymerase chain reaction (PCR) inside POPC or POPC/PS liposomes." In the former case, EDTA was added after the formation of the liposomes to inhibit the non-entrapped enzymes (and the kinetics was followed after addition of the EDTA molecules), in the latter case, the non-entrapped DNA template molecules were digested by DNase I before the temperature was raised to 95°C and the polymerization started. 

The reliability of the PCR (polymerase chain reaction) is dependent on many external factors, such as false-negative results caused by uncontrolled PCR inhibitor, but also by contamination with foreign DNA. Special attention has to be given to the breakdown of the PCR by contamination through the air the contamination... 

Reverse transcriptase polymerase chain reaction Short-acting beta-adrenoceptor agonist Standardized mortality rate Single nucleotide polymorphism Serotonin and noradrenaline reuptake inhibitor Selective serotonin reuptake inhibitor Simian virus 40... 

Before the sequencing begins it is necessary to prepare a short primer that is complementary to a sequence at one end of the DNA strand to be sequenced. This may be prepared enzymatically,622 623 or by non-enzymatic synthesis. The short primer is annealed to the end of the DNA and the resulting molecule is incubated with a DNA polymerase and a mixture of the four mononucleotide triphosphates, one of which is radio-labeled in this position. Four reaction mixtures are prepared. Each mixture contains all four nucleoside triphosphates and also one of four different chain-terminating inhibitors, the most popular of which are the 2, 3 -dideoxyribonucleoside triphosphates ... 

Fig. 3.13. Diagrammatic representation of the Forward-Backward procedure. A double-stranded DNA fragment [32P] labelled (asterisk) at one 5 -end is represented at the top of the figure. DNA polymerase I and a nucleotide chain inhibitor (e.g. ddA) are added. Contaminating DNAases in the Poll preparation produce nicks, indicated by the vertical arrows. From the 3 -end created by each nick, the reaction catalysed by Poll proceeds in the 5 - to 3 -direction (Forward reaction) provided dNTPs (dG, dT, dC) are present if they are not added the reaction proceeds exonucleolytically in the 3 - to 5 -direction (Backwards). The numbered lines represent the DNA fragments which arise from the similarly numbered DNA nicks. The hypothetical DNA sequence illustrates the complementary results obtained from the Forward and Backward reactions with repeated nucleotides e.g. the sequence AA. In the Forward reaction the proximal A will be represented by a strong band and the distal A by a weak band. The converse is true for the Backward reaction. The dotted lines 4 and 5 signify those reactions which proceed 5 - 3 (Forward) in the Backwards procedure.

Vidarabine is an inhibitor of viral DNA synthesis. Cellular enzymes phosphorylate vidarabine to the triphosphate, which inhibits viral DNA polymerase activity in a manner that is competitive with deoxyadenosine triphosphate. Vidarabine triphosphate is incorporated into both cellular and viral DNA, where it may act as a chain terminator. Vidarabine triphosphate also inhibits ribonucleoside reductase, RNA polyadenylation, and 5 -adenosylhomocysteine hydrolase, an enzyme involved in transmethylation reactions. Resistant variants due to mutations in viral DNA polymerase can be selected in vitro. 

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