Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-10
2003-04-01
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S006120, C435S091100, C435S091200, C536S023100, C536S023500, C536S024300
Reexamination Certificate
active
06541204
ABSTRACT:
The present invention relates to the prevention of false positive results in nucleic acid amplification reactions and in particular to prevention using a DNase. The invention also relates to a thermolabile DNase suitable for use in such a method.
Nucleic acid amplification techniques such as polymerase chain reactions (PCR's) are one of the most powerful tools available in biotechnology, allowing preparation of a large number of copies of a target sequence from a sample containing only a small amount of nucleic acid. In the case of PCR, oligonucleotide primers complementary to their respective strands of a double stranded target sequence are added to the reaction mixture containing the target sequence and free nucleotides. Thermal cycling in the presence of a DNA polymerase results in amplification of the sequence between the primers. The ability of the amplified fragments created by the PCR process to act as templates for subsequent PCR cycles results in the rapid production of a considerable quantity of the target sequence. Even a single copy of the target sequence can yield sufficient nucleic acid to allow detection by, e.g. hybridization with a labelled probe or incorporation of a
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P labelled deoxynucleotide triphosphates into the amplified segment.
Ligase amplification reaction (LAR) also known as ligase chain reaction (LCR), like PCR, uses repetitive cycles and alternating temperature to achieve an exponential increase in the number of copies of the target sequence. In this method, DNA ligase catalyses the joining of two oligonucleotides complementary to adjacent regions of one of the target DNA strands. Two other oligonucleotides complementary to the other strand can also be ligated. After denaturation, the original template strands and the two ligated pairs can act as templates for further hybridisation and ligation.
Reverse transcript PCR (RT-PCR) is an amplification method in which the single strand RNA (ssRNA) template is reverse transcribed into a complementary single stranded DNA which is used to form double strand DNA (dsDNA) which is then subsequently amplified in the normal way as a DNA PCR product. Some enzymes are capable of producing the first DNA strand and synthesising the second strand to form dsDNA and others are specific for just one of the two steps.
The ability of these techniques to amplify minute quantities of a target sequence makes them highly susceptible to contamination by target sequences which may be carried over from previous amplification reactions in reagents, pipetting devices, laboratory surfaces, gloves or aerosolization. Aerosols can occur by disturbing a solution such as during a spill or even by disturbing the small amount of material on a container surface such as the residue on the inner surface of a cap of a plastic tube which can be aerosolized when the tube is opened. When the sample nucleic acid is being investigated for medical diagnostic or forensic reasons, the impact of false-positive results caused by the accidental introduction into the reaction mixture of nucleic acid which may comprise the target sequence, known as carry-over, can be far-reaching.
A number of techniques for preventing or limiting the effects of carry-over have been developed. These include nested primers, primers which anneal to the target sequence inside the annealing boundaries of the two primers used to start PCR (K. B. Mullis et al. Cold Spring Harbour Symposia Vol. LI, pp 263-273, 1986). The shorter PCR amplified product of the nested primers cannot anneal with the starting primers so if it is this product which is carried over, the use of the starting primers will not amplify this carry-over. However, the carry-over has not been removed and if the same nested primers are used in a subsequent PCR, the previously amplified product of the nested primers will be amplified.
Recently, methods have been developed which involve incorporation of the nucleotide deoxyuridine triphosphate (dUTP) into amplified nucleic acid sequences in place of thymidine triphosphate (TTP). As deoxyuridine (dU) is not normally found in naturally-occurring DNA, this nucleotide distinguishes previously produced amplicons from new target sequences. Prior to the commencement of a further amplification reaction, the amplification reaction mixture can be treated with the enzyme uracil DNA glycosylase (UDG) which removes the uracil base, leaving the sugar-phosphodiester backbone intact producing an abasic site in single strand (ss) and double strand (ds) DNA (U.S. Pat. No. 5,418,149). The temperature of the amplification reaction mixture is elevated to cleave the DNA at the abasic sites which results in degradation of the carry-over. This method too is not without problems, as the introduction of dUTP in the amplification product can interfere with subsequent analysis of the product e.g. by restriction enzyme cleavage. Also, the UDG is not irreversibly inactivated at high temperatures. The temperature steps used in the amplification reaction must be above 54° C. and the reaction vessel must be kept at high temperatures or immediately frozen, to prevent the newly produced amplifications which will also contain uracil from being degraded.
It has also been suggested that individual reaction mixtures be treated prior to addition of the target DNA and Taq DNA polymerase with DNaseI or restriction endonucleases that cut internal to the pair of amplification primers thus preventing amplification of contaminating DNA (Furrer et al. Nature. Vol. 346 page 324, 1990). This method requires a decontamination time of 30 minutes and in order to inactivate the DNaseI or restriction endonuclease after decontamination, the reaction mixture is boiled. Because of this boiling step, it is necessary to add the Taq DNA polymerase after decontamination which represents a further risk of the introduction of carry-over into the pre-amplification mixture. Primer concentrations of 1 &mgr;M are used in this method.
There thus remains a need for a method which can simply and efficiently prevent false positive results due to carry-over in nucleic acid amplification reactions.
A new DNase ie. DNA-degrading enzyme, has been isolated and purified which has been found by the inventors to exhibit characteristics which make it suitable for use in the elimination or reduction of carry-over. In particular, the DNase is thermolabile, being irreversibly inactivated at high temperatures. As with all DNases, the thermolabile DNase of the invention digests dsDNA by cleaving the phosphodiester links of the sugar phosphate nucleic acid backbone.
Thus, according to the present invention, there is provided a method of removing nucleic acid contamination in an amplification reaction which comprises use of a thermolabile DNase.
The thermolabile DNase is thus used to degrade double stranded non-target DNA present in the amplification reaction mixture. Thereby, non-specific amplification may be reduced or avoided.
In particular, the method involves contacting the amplification reaction mixture with a thermolabile DNase under conditions which permit digestion of any double stranded DNA therein; heating said reaction mixture to inactivate said DNase and thereafter bringing said mixture into contact with said target nucleic acid to be amplified. The target nucleic acid (ie. the template for amplification) may simply be added to the mixture or a barrier separating the template from the remainder of the amplification reaction, removed.
Alternatively viewed, this aspect of the invention provides use of a thermolabile DNase in removing nucleic acid contamination in an amplification reaction mixture.
As mentioned above, the invention has particular utility in preventing or limiting carry-over, and in particular in preventing or reducing false positive results due to carry-over.
In a further aspect the invention also provides a method of preventing or reducing false positive results due to carry-over in nucleic acid amplification reactions, said method comprising using a thermolabile DNase to degrade carried-over non-target double-str
Nilsen Inge Waller
Sandsdalen Erling
Stenberg Even
Dorsey & Whitney LLP
Norwegian Institute of Fisheries & Aquaculture Ltd.
Taylor Janell E
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