Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-10-01
2001-03-06
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S005000
Reexamination Certificate
active
06197510
ABSTRACT:
FIELD OF THE INVENTION
The invention is in the field of nucleic acid analysis methods, including methods useful for sequence analysis and for diagnostic differentiation of cell types, such as identification of pathological organisms.
BACKGROUND OF THE INVENTION
A variety of methods are known for sequencing nucleic acids. Most DNA sequencing methods in current use are derived from the Sanger dideoxy chain-termination method (Sanger, F., S. Nicklen and A. R. Coulson (1977) “DNA sequencing with chain-terminating inhibitors” PNAS USA 74:5463-5467). These methods initiate polymerase catalysed duplication from a labelled primer complementary to a portion of the strand to be sequenced. Typically, four polymerase reactions are carried out, each with one of the four dideoxynucleotides (ddNTPs) mixed with the normal deoxynucleotides. The ddNTPs can not form a phosphodiester bond with subsequent nucleotides, so that in each reaction mixture chain polymerization is occasionally terminated at a ddNTP, producing a series of labelled strands whose lengths are indicative of the location of a particular base in the sequence. The resultant labelled fragments may be separated according to size by polyacrylamide gel electrophoresis. The position of the fragments in the gel may be determined by detecting the label, autoradiography may for example be used to detect radio-labelled fragments. Variations of the Sanger sequencing method have recently been adapted for large-scale automated sequencing using multiple fluorescent labels and capillary gel electrophoresis.
The polymerase chain reaction (PCR) may be used to amplify sequences prior to sequencing. Aspects of the PCR process are disclosed in the following United States patents: which are incorporated herein by reference: U.S. Pat. No. 4,683,195 issued Jul. 28, 1987 to Mullis et al. and U.S. Pat. No. 4,683,202 issued Jul. 28, 1987 to Mullis (see also U.S. Pat. No. 4,965,188; Saiki et al., (1988) Science 239:487-491; and, Mullis, K. B. et al. (eds.), 1994, “The Polymerase Chain Reaction”, Springer Verlag, ISBN 0817637508). The PCR makes use of primers that anneal to opposite strands at either end of an intervening sequence in order to amplify the intervening sequence. Polymerase chain reaction mixtures are heated to separate complimentary strands between cycles of active polymerization. Under appropriate conditions, such cycles may result in a million-fold amplification of the target sequence. The amplified DNA may be then sequenced.
Nucleic acid amplification techniques may usefully be employed to detect a wide variety of pathogens or other organisms. Similarly, amplification of variable regions of a genome may be used to distinguish between one organism and another, a process sometimes called DNA fingerprinting. It is possible, however, to obtain a false-positive result from amplification reactions when non-specific amplification occurs. One approach to ameliorating this problem has been the use of multiple amplification reactions on a single sample, sometimes known as multiplex amplification, as for example disclosed in U.S. Pat. No. 5,582,989 issued Dec. 10, 1996 to Caskey et al., U.S. Pat. No. 5,552,283 issued to Diamandis et al. Sep. 3, 1996 and U.S. Pat. No. 5,403,707 issued Apr. 4, 1995 to Atwood et al. U.S (all of which are incorporated herein by reference). U.S. Pat. No. 5,582,989 teaches that the original PCR methods disclosed in the above-referenced patents to Mullis and Mullis et al. are not suitable for simultaneous multiplex amplification reactions. U.S. Pat. No. 5,403,707 teaches that it is useful in multiplexed amplification reactions to use primers that are very similar in length and therefore have similar melting temperatures.
Nucleic acid amplification techniques may be adapted and combined with sequencing reaction chemistry to provide sequence information. Such a system may be called ‘cycle sequencing’, and typically involves the use of a pair of primers, analogous to amplification primers, which anneal to opposite strands at either end of a sequence of interest. The usual amplification reaction chemistry is modified by including a chain-terminating nucleotide (such as a ddNTP) in the reaction mixture, so that some of the primer extension products will terminate at the places in the sequence of interest where that nucleotide occurs. However, some of the primer extension products will reach the opposite priming site, so that they may serve as the template for primer extension in the next round of amplification. In an adaptation of this procedure, simultaneous bi-directional sequencing of both strands of a double-stranded DNA may be performed using two strand-specific primers, each carrying a unique label. A variety of commercially available kits are available that provide appropriate reagents and instructions for carrying out such reactions using thermostable polymerase: SequiTherm Long-Read Cycle Sequencing Kit-LC (Cat # S3610OLC), Epicentre Technologies, Madison, Wis., U.S.A.; SequiTherm Excel Long-Read DNA Sequencing Kit-LC (Cat. # SE610OLC), epicentre Technologies, Madison, Wis., U.S.A.; Thermo Sequenase fluorescent labelled primer cycle sequencing kit with 7-deaza-dGTP (Cat. #RPN 2438), Amersham Life Science, Cleveland, Ohio, U.S.A.; and, Circum Vent Thermal Cycle Sequencing Kit (Cat # 430-100), New England Biolabs, Beverly, Mass., U.S.A.
Mucosal disease (MD) is one of the most common viral diseases in cattle, causing significant economic loss. MD is characterized by fever, salivation, nasal discharge, diarrhoea, anorexia, dehydration, and abortion. The disease is caused by an RNA virus known as the bovine viral diarrhoea virus (BVDV). The sequences of several variants of the BVDV genome are known (Collett, M. S. et al., 1988, “Molecular Cloning and Nucleotide Sequence of the Pestivirus Bovine Diarrhoea Virus”, Virology 165: 191-199; Pellerin et al., 1994, “Identification of a New Group of Bovine Viral Diarrhoea Virus Strains Associated with Severe Outbreaks and High Mortalities”, Virolgogy 203: 260-268). BVDV belongs to a family of pestivirus which shares many similarities with viruses causing boarder disease and hog cholera. BVDV occurs in both non-cytopathogenic (ncp) and cytopathogenic (cp) strains. The ncp strain survives in animal tissues without any disruption as a latent invention. The cp strain causes cellular disruption and disease.
As a polymorphic RNA virus, BVDV is an example of the wide range of organisms for which reliable diagnostic protocols are required, and for which it would be desirable to have techniques for efficiently assaying polymorphisims that may be indicative of divergent pathologies associated with different strains of the organism. Efficient techniques for determining the evolutionary lineage of a particular pathogen, as evidenced by its complete or partial nucleic acid sequence, may also be useful in providing epidemiological information about the organism.
SUMMARY OF THE INVENTION
An object of the present invention is to provide methods for simultaneously analysing multiple nucleic acid regions in a single reaction. The methods may for example be adapted to analyse the sequences obtained from multiplexed amplification reactions. Such information may be useful for the reliable diagnosis and differentiation of pathological organisms. Such information may also be useful in genomic differentiation of individual by procedures commonly referred to as ‘DNA fingerprinting’.
In one embodiment, the invention provides a method of sequencing a nucleic acid in a reaction mixture comprising first and second nucleic acid target sequences. The target sequences may be present on the same or different nucleic acid molecules. First and second labelled sequencing primers are provided that are hybridizable, respectively, to the first and second nucleic acid target sequences. The second sequencing primer being at least as long as the total length of the first sequencing primer plus the length of the first target sequence. The reaction mixture is provided with a DNA polymerase, deoxyribonucleotid
Bio-Id Diagnostic Inc.
Lundgren Jeffrey S.
Marschel Ardin H.
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