Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-08-20
2001-06-19
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S023500, C536S023600
Reexamination Certificate
active
06248522
ABSTRACT:
The invention concerns methods for amplifying nucleic acids, methods for detecting nucleic acids with the aid of these amplification methods, the use of lauroyl sarcosine to prevent the reactivation of uracil-N-glycosilase as well as a reagent and a reagent kit for carrying out this method.
Since the introduction of methods for amplifying nucleic acids (e.g. the polymerase chain reaction (PCR) as described in U.S. Pat. No. 4,683,202) into nucleic acid diagnostics, a whole series of analytes have become analytically detectable for the first time. These methods are even able to amplify analyte nucleic acids, e.g. of HCV, that are present in the smallest concentrations to such an extent that they become accessible to those nucleic acid tests which have previously been restricted to highly concentrated analytes. However, over time it has turned out that the laboratories in which the amplifications were carried out have in the meantime already become so strongly contaminated with the amplified nucleic acids that tests in samples which in fact do not contain the low concentrated nucleic acid at all lead to false-positive results since the samples have become contaminated by the environment with nucleic acids from previous amplifications (cross-contaminations). The high sensitivity of the amplification-based nucleic acid tests enables the detection of even the slightest contaminations and hence simulates the presence of the analyte in the sample (false-positive results).
A method for stabilizing alkaline solutions containing nucleic acids with anionic, non-ionic and zwitterionic detergents is described in EP-B-0 566 050.
EP-A-0 401 037 describes a method which partially remedies the described deficiency. In this method mononucleotides that are not naturally present in the nucleic acid to be detected are incorporated during the amplification into the amplificate of each analyte nucleic acid. Before a subsequent amplification is carried out, the sample together with the reagents used are subjected to a pretreatment in which all imported amplificates from earlier amplifications are enzymatically degraded. Uracil-N-glycosilase (UNG) is an example of a degradation reagent and dUTP is an example of a modified building block for the amplificates. An alternative method utilizes primers containing uracil instead of mononucleotides containing uracil. Such a method in which the primer binding sites are degraded on amplificates generated earlier is described in EP-A-0 415 755.
The mechanism of this decontamination method is based on the specific recognition of uracil-containing amplificates which are degraded by the enzyme. In the preparation of the amplification reaction UNG is added to the sample and usually already together with the master mix which contains all reagents necessary for the amplification. The aforementioned degradation reaction takes place in a brief incubation step before the subsequent amplification. If the reaction mixture is subsequently heated to a temperature above ca. 40° C., then UNG is inactivated. This is necessary to ensure that the UNG does not degrade the newly synthesized DNA which accumulates during the course of the amplification.
If, however, the detection reaction does not directly follow the amplification, the denatured enzyme refolds and hence regains its activity (BioTechniques 13, 181-183, 1992). This can lead to false-negative test results. Various methods have previously been used to prevent the UNG reactivation. In a first variant the amplificates are stored at higher temperatures (e.g. >50° C.). However, this requires either an additional thermostated instrument or the thermocycler position is blocked by the reaction vessel until the test is continued. On the other hand a temperature-dependent degradation of the amplificate also occurs over time under these conditions. In a further variant the amplificates are stored at low temperatures (e.g. 4° C.). This variant also leads to additional hardware requirements and thus to increased costs. In a third variant a stop solution (usually NaOH) is added to the reaction solution after carrying out the amplification. However, adding a stop solution by pipette reduces convenience for the operator and is invariably linked with an opening of the reaction tube whether this is carried out manually or automatically. However, the risk of contamination also considerably increases with each additional working step especially after amplification.
Therefore the object of the present invention was to completely or partially avoid the disadvantages of the prior art and in particular to provide a more simple amplification method with a reduced risk of contamination.
The invention concerns a method for amplifying nucleic acids in a sample comprising the steps
treating the sample with an enzyme under conditions in which products from the amplifications of nucleic acids of other samples are enzymatically degraded,
inactivating the enzyme and
treating the sample under conditions in which the nucleic acids are amplified,
wherein the amplification is carried out in the presence of a reagent that prevents reactivation of the enzyme.
The invention also concerns a reagent that can be used in the above method and the use of detergents in methods for the amplification of nucleic acids.
REFERENCES:
patent: 5418149 (1995-05-01), Gelfland et al.
Haberhausen Gerd
Jäger Stephan
Sobek Harald
Arent Fox Kintner Plotkin & Kahn
Horlick Kenneth R.
Roche Diagnostics GmbH
Taylor Janell E.
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