Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-14
2004-12-14
Forman, BJ (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06830884
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to a method of amplifying closed circular nucleic acid probes and, more particularly, to a method of amplifying closed circular nucleic acid probes by rolling circle amplification. The method of the present invention is useful in a range of applications involving the detection of nucleic acid sequences such as, but not limited to, the identification of genetic disorders, genetic variants or the presence of microbiological or viral agents.
BACKGROUND OF THE INVENTION
Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.
A variety of nucleic acid amplification technologies exist for the diagnosis of infectious and genetic diseases. Since its invention over a decade ago, the polymerase chain reaction (PCR) (1) has become the method of choice in research and DNA-based diagnostics. This can be attributed to its speed, simplicity and sensitivity. PCR does, however, require temperature cycling, which therefore necessitates the use of expensive thermal cycling equipment. Other amplification techniques, which also require temperature cycling, include the ligase chain reaction (LCR) (2) and the transcription-based amplification system (TAS) (3).
Various other amplification techniques exist which do not require extensive thermal cycling and are essentially isothermal systems. Several of these are transcription-medited or require RNA as an integral component of the reaction therefore necessitating that the amplification environment is kept free from ribonuclease contamination. These methods include the Q&bgr; replicase system (4), self-sustained sequence replication (3SR) (5) and nucleic acid sequence-based amplification (NASBA) (6).
Presently, there appear to exist at least two isothermal techniques for the amplification of nucleic acid sequences which essentially do not require RNA intermediates. Strand displacement amplification (SDA) (7) is an isothermal technique which relies on the ability of a restriction enzyme to nick a hemiphosphorothioated recognition site and the ability of a polymerase to initiate replication at a nick and displace the downstream strand. The other isothermal technique which can be used to amplify a nucleic acid sequence is rolling circle amplification (RCA).
Various forms of the rolling circle amplification technique have previously been described (8,9). In essence the technique relies on amplification from a circular DNA probe. The circular probe, commonly referred to as a “padlock probe”, is designed such that it has regions at both its 5′ and 3′ ends which are complementary to the target sequence of interest and are separated by a region of nucleotide of non-target derived origin. Upon hybridisation, the 5′ and 3′ ends of the probe are brought into ciose proximity to one another. If the two probe regions are adjacent to one another the 5′ and 3′ ends can be joined to produce a circular probe, In some instances, however, the probe regions are separated from one another by a small stretch of nucleotides. This region must be filled to achieve the generation of a circular probe. In this regard, a variety of techniques can be utilised including the use of spacer oligonucleotides or by using a DNA polymerase (or a reverse transcripts in the case of an RNA target) in combination with deoxynucleotide triphosphate molecules to fill the gap prior to ligation.
A significant problem associated with the rolling circle amplification technique is the occurrence of background amplification. Prior to the advent of the present invention this background amplification was dismissed as primer-induced deletion fragment repeats encompassing a full unit repeat minus the intervening region between 5′ ends of the two primers (8). Background amplification represents both a significant problem and a limitation for rolling circle amplification reactions which utilise 2 primers. It is also a major source of false positive results. In fact, the magnitude of the problem presented by the occurrence of this background amplification bas been such that it has not been feasible to use the two primer rolling circle amplification techniques with an acceptable level of specificity.
In work leading up to the present invention the inventors have determined the origin of and characterised this background amplification. This class of background amplification has been termed “AmpX”. The inventors have determined that it is an alternative amplification reaction which utilizes any linear nucleic acid probe molecules present in the reaction mixture. Typically the reaction products are multimers of head to tail tandem repeats. However, the inventors have determined that rather than encompassing sequence from the entire circular probe, the products of the AmpX reaction include repeats of a region of the linear target molecule that includes the two primer binding sites, the intervening sequence and some additional sequence of the template molecule flanking the primer binding sites.
Accordingly, the inventors have developed a method for minimizing AmpX background amplification by enriching for closed circular nucleic acid probe molecules prior to their amplification. By conducting the amplification step utilising an enriched population of closed circle nucleic acid probe molecules the incidence of background amplification caused by the AmpX reaction is significantly reduced, thereby enabling more specific rolling circle amplification to occur.
SUMMARY OF THE INVENTION
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
The subject specification contains nucleotide sequence information prepared using the programme PatentIn Version 2.0, presented herein after the bibliography. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1, <210>2, etc). The length, type of sequence (DNA, etc) and source organism for each nucleotide sequence are indicated by information provided in the numeric indicator fields <211>, <212>and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (e.g. <4001>1, <400>2, etc).
Accordingly, one aspect of the present invention provides a method for amplifying a circular nucleic acid probe produced following interaction of a nucleic acid probe with a target nucleic acid sequence said method comprising enriching said circular nucleic acid probe and subjecting said circular nucleic acid probe to amplification.
Another aspect of the present invention provides a method of rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
Still another aspect of the present invention more particularly provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
A further aspect of the present invention provides a method for amplifying a circular nucleic acid probe produced following interaction of a nucleic acid probe with a target nucleic acid sequence said method comprising enzymatically enriching for said circular nucleic acid
Dale James Langham
Giffard Philip Morrison
Hafner Gregory John
Stafford Mark Richard
Voisey Joanne
Forman BJ
Molecular Staging Inc.
Needle & Rosenberg P.C.
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