Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-05-16
2001-11-06
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091200, C435S005000, C435S006120
Reexamination Certificate
active
06312928
ABSTRACT:
The present invention is directed to a transcription based amplification method for the amplification of DNA targets.
Nucleic acid amplification methods are used in the field of molecular biology and recombinant DNA technology. These methods are used to increase the number of copies of a particular nucleic acid sequence, present in small amounts and often in an environment in which a wide variety of other nucleic acid sequences, both RNA and DNA, are also present. In particular, nucleic acid amplification methods are used to facilitate the detection or quantification of nucleic acid and are important for diagnosing for example infectious disenses, inhereted dieseases and various types of cancer. Nucleic acid amplification methods have also found their applications in other fields where samples are investigated in which nucleic acid may be present in minute amounts, such as forensic sciences, archeology or to establish paternity.
Several nucleic add amplification techniques are known based on different mechanisms of action. One method for the amplification of nucleic acid is known as the “Polymerase Chain Reaction” (PCR) is described in European patent applications EP 200362 and EP 201148. PCR is a cyclic process which has double stranded DNA as target. Each cycle in the PCR process starts with the separation of a double stranded DNA target in its two complementary strands. To each strand a primer will anneal and DNA polymerases present will extend the primers along the DNA strand to which it annealed thus forming two new DNA duplexes. When the reaction mixture is heated the strands of the DNA duplexes will be separated again and a new PCR cycle can start. Thus, the PCR process produces multiple DNA copies of a DNA target. If single stranded RNA is the desired target for PCR, it has to be converted to double stranded DNA first by reverse transcriptase.
The present invention is concerned with a different class of nucleic acid amplification methods namely the transcription based amplification techniques. The techniques involve the transcription of multiple RNA copies from a template comprising a promoter recognized by an RNA polymerase. With these methods multiple RNA copies are transcribed from a DNA template that comprises a functional promoter recognized by the RNA polymerase. Said copies are used as a target again from which a new amount of the DNA template is obtained etc. Such methods have been described by Gingeras et al. in WO88/10315 and Burg et al. in WO89/1050. Isothermal transcription based amplification techniques have been described by Davey et al. in EP 323822 (relating to the NASBA method), by Gingeras et al. in EP 373960 and by Kacian et al. in EP 408295. Transcription based amplification reactions may also be performed with thermostable enzymes. Transcription based amplifications are usually carried out at a temperature around 41 degrees Celsius. These thermostable enzymes allow the reaction to be carried out at more elevated temperatures. Such a thermostable method is described in EP 682121 filed in the name of Toyo Boseki KK. The methods as described in EP 323822, EP 373960 and EP 408295 are isothermal continuous methods. With these methods four enzyme activities are required to achieve amplification: an RNA dependent DNA polymerase activity, an DNA dependent DNA polymerase activity, an RNase (H) activity and an RNA polymerase activity. Some of these activities can be combined in one enzyme, so usually only 2 or 3 enzymes are necessary. Enzymes having RNA dependent DNA polymerase activities are enzymes that synthesize DNA from an RNA template. A DNA dependent DNA polymerase thus synthesizes DNA from a DNA template. In transcription based amplification reactions a reverse transcriptase such as AMV (Avian Myoblastosis Virus) or MMLV (Moloney Murine Leukemia Virus) reverse transcriptase may be used. Such enzymes have both RNA- and DNA dependent DNA polymerase activity but also an inherent RNase activity. In addition an RNase may be added to the reaction mixture of a transcription based amplification reaction, such as
E. coli
RNase H.
DNA dependent RNA polymerases synthesize multiple RNA copies from a DNA template including a promoter recognized by the RNA polymerase. Examples of RNA polymerases are polymerases from
E. coli
and bacteriophages T7, T3 and SP6. An example of an RNA polymerase commonly used with transcription based amplification methods is T7 polymerase. Thus the promoter that is incorporated in the template used for transcribing multiple copies of RNA would then be the T7-promoter. Usually the template comprising the promoter has to be created starting from the nucleic acid comprising the target sequence. Said nucleic acid may be present in the starting material that is used as input for the amplification reaction. The nucleic acid present in the starting material will usually contain the target sequence as a part of a much longer sequence. Additional nucleic acid sequences may be present on both the 3′- and the 5′-end of the target sequence. The amplification reaction can be started by bringing together this nucleic acid from the starting material, the appropriate enzymes that together provide the above mentioned activities and at least one, but usually two, oligonucleotide(s). At least one of these oligonucleotides should comprise the sequence of the promoter.
Transcription based amplification methods are particularly useful if the input material is single stranded RNA, although single or double stranded DNA can likewise be used as input material. When a transcription based amplification method is practiced on a sample with single stranded RNA (of the “plus” sense) with additional sequences on both the 3′-end and the 5′ end of the target sequence a pair of oligonucleotides that is conveniently used with the methods as described in the prior art would consist of:
a first oligonucleotide (usually referred to a “promoter-oligonucleotide”) that is capable of hybridizing to the 3-end of the target sequence, which oligonucleotide has the sequence of a promoter (preferably the T7 promoter) attached to its 5 ′ end (the hybridizing part of this oligonucleotide has the opposite polarity as the plus RNA used as input material).
a second oligonucleotide (“primer”) which comprises the 3′ end of the target sequence (this oligonucleotide has the same polarity as the plus RNA).
When such a pair of oligonucleotides, together with all enzymes having the appropriate activities, and a sufficient supply of the necessary ribonucleotides and deoxy-ribonucleotides are put together in one reaction mixture and are kept under the appropriate conditions (that is, under the appropriate buffer conditions and at the appropriate temperature) for a sufficient period of time an isothermal continuous amplification reaction will start.
FIG. 1
gives a proposed mechanism for part of a transcription based amplification reaction known in the art. The isothermal continuous process of amplification is pictured in
FIG. 1
as a cyclic process. However, in fact all steps of this process will take place at the same time since all ingredients are present in the reaction vessel. Thus, picturing the process as a particular sequence of events might be good for a better understanding of a possible mechanism underlying the amplification process, it might not be a real reflection of what is actually happening in the reaction mixture during amplification. The cycle depicted in
FIG. 1
can be regarded as starting with a first amount of single stranded RNA. The RNA depicted in the cycle is RNA of the minus sense. Thus, it will be able to hybridize with the second oligonucleotide of the pair of oligonucleotides mentioned above. This minus RNA will normally not be present as such in the starting material for the amplification reaction but will be derived from, for example, the plus RNA present in the starting material through reaction with the oligonucleotides and enzymes when all ingredients of the reaction mixture have been brought together.
Many variants of
Schukkink Adriana Fredericke
Van Gemen Bob
Van Strijp Dianne Arnoldina Margaretha
Akzo Nobel N.V.
Jones W. Gary
Sullivan Michael G.
Taylor Janell
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