Use of nucleic acid analogues in the inhibition of nucleic acid

Details Use of nucleic acid analogues in the inhibition of nucleic acid Use of nucleic acid analogues in the inhibition of nucleic acid

1995-03-10

1999-04-06

Jones, W. Gary

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 911, 435 912, 536 245, 935 77, 935 78, C12Q 168, C12P 1934, C07H 2104, C07H 2102

Patent

active

058916253

DESCRIPTION:

BRIEF SUMMARY
This application is a 371 of the International Application PCT/EP93/01435, filed Jun. 7, 1993.


BACKGROUND OF THE INVENTION

The present invention relates to the use of nucleic acid analogues in blocking nucleic acid amplification procedures and to diagnostic/analytical techniques based thereon.
Nucleic acid amplification techniques are now in widespread use. These include the "PCR" (polymerase chain reaction) procedures described in EP-A-0200362 and EP-A-0201184 which is the technique in most widespread use, but also the "LCR" (ligase chain reaction) described in EP-A-0320308 and the so-called "NASBA" or "3SR" technique which is described in "Proc. Natl. Acad. Sci. USA" Vol 87. pp 1874-1878 March 1990 and "Nature" Vol 350, No 6313. pp 91-92 7th March 1991.
A major problem in the use of these procedures in diagnostics is the production of false positives by the carry over of amplified nucleic acid sequences from previous reactions. As these procedures are capable of producing a positive result if even a single molecule containing the target sequence is present, it is of course very easy for such a false positive to occur. At present it is necessary for the reaction product of such procedures to be worked up before the success or failure of the amplification procedure can be determined. The involves contact between the reaction product and several pieces of laboratory equipment such as pipettes, as well as with personnel which can lead to traces of the amplification product being available to contaminate future runs.
Much effort is currently going into developing so called "intrinsic" procedures in which the success of the amplification reaction can be monitored without any handling of the amplification product and in an unopened reaction vessel.
New forms of nucleic acid analogue are described in Patent Co-operation Treaty application. No. PCT/EP92/01220 filed on 22nd May 1992 which selectively bind conventional nucleic acids of complementary sequence to form hybrids which are more stable against dehybridisation by heat than are similar hybrids between conventional nucleic acids. We have now found that it is possible to exploit this greater hybrid stability to block selectively nucleic acid amplification procedures. This technique may be used to prevent false positives in such amplification procedures. It may also be exploited as an essential part of certain diagnostics/analytical approaches.
In a first aspect the invention provides a method of inhibiting a nucleic acid amplification procedure comprising providing in said procedure an effective amount of a nucleic acid analogue sufficiently complementary to a sequence of nucleic acid participating in an essential manner in said amplification procedure to hybridises to said sequence sufficiently strongly to prevent the effective participation of said sequence in the amplification procedure. Thus this aspect of the invention includes a method of inhibiting a nucleic acid amplification procedure in which procedure each strand of a double stranded target nucleic acid has a region used as a template for one or more primers which is or are extended or linked to form a second template complementary to said template, wherein a nucleic acid analogue sufficiently complementary to a sequence of a said template or a said primer to hybridise therewith is provided and wherein said nucleic acid analogue hybridises sufficiently strongly to the respective template or primer to block primer template hybridisation or to block primer extension or primer linking under the conditions of the procedure.
Preferably, said procedure is a PCR or LCR or a 3SR procedure. "Anchored" and "inverted" PCR procedures are included.
The "double stranded target nucleic acid" which has in each strand a region acting as a template is not necessarily a starting material, nor need it necessarily be the direct object of desired amplification in the procedure.
In PCR the "double stranded target nucleic acid" will usually be incorporated in the starting material and it will generally be the amplifica

REFERENCES:
patent: 4683195 (1987-07-01), Mullis et al.
patent: 4988617 (1991-01-01), Landegren et al.
Neilsen et al., Science 254:1497-1500, 1991.
Mier et al., Angen.Chem. Int. Ed. Engl. 31(8):1008-1011, 1992.

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