Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-03-30
2002-06-11
Whisenant, Ethan C. (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C436S094000, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06403340
ABSTRACT:
The present invention concerns novel methods for detecting single-stranded target nucleic acid sequences.
As used herein the term “nucleic acid” includes protein nucleic acid (i.e. nucleic acids in which the bases are linked by a polypeptide backbone) as well as nucleic acids (e.g. DNA and RNA) having a sugar phosphate backbone, and chemical analogues thereof.
Generally speaking, amplification techniques may be viewed as effecting the detection of a target by amplifying it. However, these typically require a large target sequence since it is to be amplified and must be unambiguously identifiable in order to reduce background noise. These techniques also amplify only the detected sequence, and are unable to provide separate distinct sequences for subsequent detection, for example sequences which will hybridise to other sequences in a detection system, or which are specifically bound by antibodies. For example, amplification techniques used to detect different targets are unable to generate a common product for all of the different targets. Amplification techniques also typically require the use of thermal cycling, thus causing the reaction to pass through many heating and cooling steps. This slows down the reaction, requires the use of expensive thermal cycler devices and of larger quantities of reagents or the use of more expensive thermostable reagents.
One example of an amplification technology is PCR (EP 0201184) in which a pair of complementary template strands are treated with a molar excess of two oligonucleotide primers so that the primers hybridize to the template strands. A polymerase enzyme is then used to extend the primers to produce strands complementary to the template strand sequences. The hybridised templates and complementary strands are then separated by heat denaturation to give two new pairs of complementary template strands. Subsequent cooling allows further primers to hybridise to the template strands. By repeating the cycle of steps, an exponential amplification of the complementary template strands is achieved. A particular disadvantage encountered with PCR is the need to cycle the reaction through many heating and cooling steps, as discussed above.
Isothermal amplification procedures are also known which similarly use a molar excess of primers but avoid the need for thermal cycling by using strand displacing polymerasc enzymes (i.e. polymerase enzymes lacking 5′ to 3′ exonuclease activity), which displace the non-template part of any double stranded DNA they encounter whilst themselves progressing along a template DNA strand to produce double stranded DNA.
Such isothermal amplification techniques are exemplified by the Strand Displacement Amplification of EP 0497272, which provides for amplification of a target nucleic acid sequence, but requires a pair of primers, at least one template strand, and the presence of at least one substituted inter-nucleotide linkages, meaning that amplification products all contain substituted deoxynucleosidetriphosphates. It also requires the use of a second set of primers in reactions.
Rolling Circle Amplification is another isothermal amplification system, requiring a circular single-stranded template. Primers hybridised to the template allow the binding and activity of strand displacing polymerases which circle around the template displacing non-template sequence, and which produce just one single stranded complementary strand containing a number of tandem repeats of the sequence complementary to the template strand. However, the unit copies within the concatamer cannot be manipulated further by processes such as extension against another template unless they have undergone additional further processing. As such they are of extremely limited use when compared to the multiple fixed-length linear products of PCR amplification.
Rolling Circle Amplification can achieve exponential amplification by including a second primer which is complementary to the concatameric strand produced by the first primer. In a cascade reaction the second primer hybridises to the concatameric strand as it is produced and the polymerase enzyme extends it (using the concatamer as template) to generate product. As the linear amplification reaction occurs further sites for hybridisation of the second primer are produced upstream of the extended product. Extension of additional second primer from these sites leads to displacement of the downstream non-template strands which in turn reveal sites for hybridisation of further first primer sequences. Thus occurs a cascade of exponential production of sites and hybridisation thereto and polymerisation of primers therefrom. A disadvantage of this method is the requirement for some pre-treatment of the target (e.g. a ligation of a synthetic nucleic acid, against the target to form a circle) and three synthetic molecules are involved in the most beneficial embodiments of the invention.
The Q&bgr; replicase amplification system relies on the target sequence to anchor a modified version of midivariant 1 (MDV1) to a solid phase, while non-anchored sequences are removed by washing. MDV1 is an RNA molecule which is the specific target for amplification by the RNA-directed RNA polymerase (replicase) of bacteriophage Q&bgr;, producing up to 10
6
to 10
9
copies of MDV1 in 15 minutes. An inherent problem with the system is the need for scrupulous w ashing to remove solution phase copies of MDV1 which are, otherwise, amplified to the same extent as target bound copies, producing a high level of background “noise”.
Cycling probe amplification utilises a chemically synthesised probe containing linkages, between some of its nucleotides, which (only when the molecule is rendered double stranded by hybridisation with the target) can be broken by the action of an enzyme. In a typical reaction, the target is denatured with the probe and the reaction is then held at a temperature at which intact probe molecules will hybridise to the target but at which the shorter products of enzymatic degradation will not. Thus, probe molecules hybridise, are cleaved by the enzyme, and the shorter products are displaced by the hybridisation of new probe molecules, in a cyclic reaction. The reaction is essentially linear in nature with the reaction products currently being detected by gel electrophoresis.
U.S. Pat. Nos. 5,645,987 and 5,863,732 (which are incorporated herein by reference in their entirety) disclose the enzymatic synthesis of oligonucleotides using a template strand having a priming region, intervening region and complementary region having a cutting attenuation modification. The priming region is complementary to a desired target oligonucleotide sequence. Upon hybridisation of the target sequence to the priming region, the target sequence is extended by polymerase activity to provide a complementary strand. This results in the production of a double stranded restriction endonuclease recognition sequence which is nicked by the restriction endonuclease, the template strand not being cut because of the effect of the cutting attenuation modification. Additional polymerase activity extends the 3′ terminus of the nicked complementary strand 5′ to the restriction site, displacing the complementary strand 3′ to the cleavage site (i.e. complementary to the complementary region of the template strand) and creating another double-stranded restriction endonuclease recognition sequence. The process can continue ad infinitum to give a linear rate of synthesis of oligonucleotides complimentary to the template strand complimentary region.
A cascade of such reactions can be employed to give non-linear rates of amplification, the displaced complimentary strand from a first reaction acting as a target oligonucleotide complimentary to the priming region of a second template strand, and so on.
WO 92/01813 and Terrance Walker, G. et al. (1992, PNAS USA, 89(1): 392-396; PMID 1309614) disclose methods of circular extension and of amplification.
No suggestion is made as to how to achieve non-linear rates of amplifica
Lu Frank
Tepnel Medical Limited
Wallenstein & Wagner Ltd.
Whisenant Ethan C.
LandOfFree
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