Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-09-19
1999-11-16
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 911, 435 912, 4352872, 422 681, C12Q 168, C12P 1934, C12M 134
Patent
active
059855550
DESCRIPTION:
BRIEF SUMMARY
The invention concerns methods for processing nucleic acids by means of a temperature regulation element as well as devices and instruments for carrying out these methods.
In analytics and especially in medical diagnostics more and more methods are beginning to be established which are based on nucleic acid tests and syntheses. Nucleic acids are for example very well suited as a very specific detection agent for organisms for the diagnosis of diseases. During these detection methods various processing steps such as denaturation, hybridization, syntheses and immobilization of nucleic acids and enzymatic treatment thereof are common. For a long time a problem of such methods was the small amount of nucleic acids in the samples.
A solution for this problem, which has made it possible to detect numerous analytes, was the amplification of nucleic acids. Such a method is described in EP-A-0 200 362 i.e. the polymerase chain reaction (PCR). In this method many copies of the original nucleic acid are produced by repeated extension of primers in a reaction solution. These can for example be detected by hybridization with a labelled nucleic acid probe according to EP-A-0 201 184. The reaction solutions used for this are heated to particular temperatures and cooled again at intervals in order to separate double strands and for the extension reaction. Since the volumes are relatively large, the times required for temperature regulation result in a relatively long period to carry out the entire amplification process.
It has been attempted to remedy this by so-called capillary PCR. In this method the reaction mixture is present in glass capillaries of a small diameter. As a result the time required to carry out an amplification sequence can be reduced to ca. 30 min. A problem with capillary PCR is the delicacy of the glass of the capillary which also has to be heated and the cumbersome sample application.
Recently more and more amplification processes are also being described in which the amplification reactions can be carried out in a closed system i.e. without supplying reagents during the cycles. Thus in a system is described WO 92/07089 in which the amplification mixture is passed as long as necessary through a circulation system in which the reaction mixture is repeatedly heated and cooled. The period in which the mixture is retained in the individual zones of the system can be influenced by selecting the diameter. An amplification process is described in EP 0511712A1 in which the amplification mixture is brought cyclically to quite specific temperatures in order to achieve relatively short cycle times. In this case also the sample handling is made more difficult.
The object of the present invention was among others to improve the existing nucleic acid processing methods and in particular to provide processes in which the amplification can be completed in a particularly short time.
The invention therefore concerns a method for processing and in particular amplifying nucleic acids in a reaction mixture characterized in that the temperature of a surface adjoining the reaction mixture and its immediate surroundings is regulated but the main space of the reaction mixture remains essentially isothermal.
The invention also concerns a device for processing and amplifying nucleic acids with a temperature regulation element as well as an instrument which contains this device.
Nucleic acids within the sense of the invention are all types of nucleic acids, modified or unmodified. Unmodified nucleic acids are for example the naturally occurring nucleic acids. Modified nucleic acids can be formed by substituting groups of the natural nucleic acids by other chemical residues. Examples are nucleic acid phosphonates or phosphothioates and nucleic acids modified on their sugar residues or bases by chemical groups which may also be detectable.
The processing of nucleic acids according to the present invention preferably includes at least one reaction step which proceeds at an increased temperature that is at least different from the
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Boehringer Mannheim GmbH
Horlick Kenneth R.
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