Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1994-02-28
1996-09-10
Zitomer, Stephanie W.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
435 6, 435 912, 435810, C12P 1934, C12Q 100, C12Q 168, C12Q 170
Patent
active
055544983
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/GB92/01599, filed Sep. 1, 1992.
BACKGROUND OF THE INVENTION
This invention concerns a new class of reagents for use in the field of molecular biology and related areas of biochemistry. The wide and general usefulness of these reagents is based upon the widespread role of the divalent cations, magnesium (Mg.sup.2+) and calcium (Ca.sup.2+) in reactions involving nucleic acids. The magnesium cation in particular affects the annealing pattern of nucleic acid strands, the secondary and tertiary structure of DNA, RNA or RNA/DNA strands, and the properties of nucleotides which universally tend to complex with divalent cations.
In addition, divalent cations are of great importance in moderating the function of a wide range of nuclease enzymes which digest nucleic acid strands into their component monomeric nucleotides and nucleosides and are also important in the function of DNA and RNA polymerase enzymes which assemble DNA and RNA strands from their component nucleotides. The rate of function of nucleic acid polymerases, the processivity (tendency to continue forward reactions along a template strand), the accuracy, and the tolerance for improper or abnormal base sequences or substitute nucleotides are all well known to be affected by variations in the concentration of magnesium in the reaction medium.
It has been known for a number of years that various other divalent cations, most particularly manganese, could substitute for magnesium and essentially replicate its effects though often at somewhat lower concentrations. However, since magnesium is a convenient and inexpensive reagent, and since none of these cation substitutions achieved any biochemically novel or helpful additional result, there has been no industrial application of such substitutions. Further, many divalent cations of transition metals cannot tolerate the thioprotectants and reductants such as .beta.-mercaptoethanol and dithiothreitol (DTT) which are required by several of the polymerase enzymes for proper function, since these reductants tend to reduce and precipitate the transition metal cations.
BRIEF SUMMARY OF THE INVENTION
The subject invention concerns a method for amplifying the polymerase activity of a nucleic acid polymerase using Group 3 ions. Advantageously, the Group 3 ions of the subject invention can be used to inhibit the activity of nucleases or other enzymes that may be present in a sample. Specifically exemplified are the Group 3 ions scandium and lanthanum. The subject invention further concerns a method for identifying an unknown nucleic acid polymerase or other enzymes in a sample.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the polymerase activity of reverse transcriptase (RT) when treated with various cations.
DETAILED DESCRIPTION OF THE INVENTION
The inventors have very surprisingly discovered, however, that when certain obscuring factors are controlled for, and when several less common cations, particularly trivalent cations are used in place of magnesium or calcium or in mixtures with them, that several novel and very useful effects can be achieved. It is helpful that these cations are not reduced by DTT or .beta.-mercaptoethanol. It has been known that some such trivalent cations could substitute for e.g. calcium in some enzymes and cause an enzyme inhibition effect. It has been demonstrated by the inventors that it is possible to inhibit nuclease enzymes in this way.
Most importantly, however, the inventors have made the surprising scientific discovery that many trivalent cations can actually increase the processivity of some nucleic acid polymerase enzymes. In the case of the reverse transcriptase enzyme which is used by some pathogenic retroviruses such as the HIV virus which causes AIDS, it is possible to increase the rate of DNA production and to increase the average length of the DNA copies of RNA templates made by the enzyme. This is most important when one wishes to assay a sample of potentially infected human blood to see if it contains any viral reverse transc
REFERENCES:
Erlich A. Henry, "PCR Technology--Principles and Applications for DNA Amplification," Published 1990 by Stockton Press (U.S.), pp. 1-22.
Sarkander, H.-I., C. G. Uthoff (1978) "Modification of RNA Synthesis in Isolated Rat Liver Nuclei during Anticoagulant Treatment with Lanthanides" Arzneimittel Forschung 28(1):21-24.
Filler Aaron G.
Lever Andrew M. L.
Syngenix Limited
Tran Paul B.
Zitomer Stephanie W.
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