Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-29
2001-10-23
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C436S094000, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06306597
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to methods for determining the nucleotide sequence of a polynucleotide, and more particularly, to a method of identifying nucleotides in a template by stepwise extension of one or more primers by successive ligations of oligonucleotide blocks.
BACKGROUND
Analysis of polynucleotides with currently available techniques provides a spectrum of information ranging from the confirmation that a test polynucleotide is the same or different than a standard or an isolated fragment to the express identification and ordering of each nucleoside of the test polynucleotide. Not only are such techniques crucial for understanding the function and control of genes and for applying many of the basic techniques of molecular biology, but they have also become increasingly important as tools in genomic analysis and a great many non-research applications, such as genetic identification, forensic analysis, genetic counselling, medical diagnostics, and the like. In these latter applications both techniques providing partial sequence information, such as fingerprinting and sequence comparisons, and techniques providing full sequence determination have been employed, e.g. Gibbs et al, Proc. Natl. Acad. Sci. 86: 1919-1923 (1989); Gyllenstein et al, Proc. Natl. Acad. Sci, 85:7652-7656 (1988); Carrano et al, Genomics, 4:129-136 (1989); Caetano-Anolles et al, Mol. Gen. Genet, 235:157-165 (1992); Brenner and Livak, Proc. Natl. Acad. Sci., 86:8902-8906 (1989); Green et al, PCR Methods and Applications, 1: 77-90 (1991); and Versalovic et al, Nucleic Acids Research, 19: 6823-6831 (1991).
Native DNA consists of two linear polymers, or strands of nucleotides. Each strand is a chain of nucleosides linked by phosphodiester bonds. The two strands are held together in an antiparallel orientation by hydrogen bonds between complementary bases of the nucleotides of the two strands: deoxyadenosine (A) pairs with thymidine (ID and deoxyguanosine (G) pairs with deoxycytidine (C).
Presently there are two basic approaches to DNA sequence determination: the dideoxy chain termination method, e.g. Sanger et al,
Proc. Natl. Acad. Sci
. 74 :5463-5467 (1977); and the chemical degradation method, e.g. Maxam et al,
Proc. Natl. Acad. Sci
. 74: 560-564 (1977). The chain termination method has been improved in several ways, and serves as the basis for all currency available automated DNA sequencing machines, e.g. Sanger et al,
J. Mol. Biol
., 143: 161-178 (1980); Schreier et al,
J. Mol. Biol
., 129: 169-172 (1979); Smith et al,
Nucleic Acids Research
, 13: 2399-2412 (1985); Smith et al,
Nature
, 321: 674-679 (1987); Prober et al,
Science
, 238: 336-341 (1987); Section II,
Meth. Enzmol
., 155: 51-334 (1987); Church et al,
Science
, 240: 185-188 (1988); Hunkapiller et al, Science, 254: 59-67 (1991); Bevan et al, PCR Methods and Applications, 1: 222-228 (1992).
Both the chain termination and chemical degradation methods require the generation of one or more sets of labeled DNA fragments, each having a common origin and each terminating with a known base. The set or sets of fragments must then be separated by size to obtain sequence information. In both methods, the DNA fragments are separated by high resolution gel electrophoresis, which must have the capacity of distinguishing very large fragments differing in size by no more than a single nucleotide. Unfortunately, this step severely limits the size of the DNA chain that can be sequenced at one time. Sequencing using these techniques can reliably accommodate a DNA chain of up to about 400-450 nucleotides, Bankier et al,
Meth. Enzymol
., 155: 51-93 (1987); and Hawkins et al, Electrophoresis, 13: 552-559 (1992).
Several significant technical problems have seriously impeded the application of such techniques to the sequencing of long target polynucleotides, e.g. in excess of 500-600 nucleotides, or to the sequencing of high volumes of many target polynucleotides. Such problems include i) the gel electrophoretic separation step which is labor intensive, is difficult to automate, and introduces an extra degree of variability in the analysis of data, e.g. band broadening due to temperature effects, compressions due to secondary structure in the DNA sequencing fragments, inhomogeneities in the separation gel, and the like; ii) nucleic acid polymerases whose properties, such as processivity, fidelity, rate of polymerization, rate of incorporation of chain terminators, and the like, are often sequence dependent; iii) detection and analysis of DNA sequencing fragments which are typically present in fmol quantities in spacially overlapping bands in a gel; iv) lower signals because the labelling moiety is distributed over the many hundred spacially separated bands rather than being concentrated in a single homogeneous phase, and v) in the case of single-lane fluorescence detection, the availability of dyes with suitable emission and absorption properties, quantum yield, and spectral resolvability, e.g. Trainor, Anal. Biochem., 62: 418-426 (1990); Connell et al, Biotechniques, 5: 342-348 (1987); Karger et al, Nucleic Acids Research, 19: 4955-4962 (1991); Fung et al, U.S. Pat. No. 4,855,225; and Nishikawa et al, Electrophoresis, 12: 623-631 (1991).
Another problem exists with current technology in the area of diagnostic sequencing. An ever widening array of disorders, susceptibilities to disorders, prognoses of disease conditions, and the like, have been correlated with the presence of particular DNA sequences, or the degree of variation (or mutation) in DNA sequences, at one or more genetic loci. Examples of such phenomena include human leukocyte antigen (HLA) typing, cystic fibrosis, tumor progression and heterogeneity, p53 proto-oncogene mutations, ras proto-oncogene mutations, and the like, e.g. Gyllensten et al, PCR Methods and Applications, 1: 91-98 (1991); Santamaria et al, International application PCT/US92/01675; Tsui et al, International application PCT/CA90/00267; and the like. A difficulty in determining DNA sequences associated with such conditions to obtain diagnostic or prognostic information is the frequent presence of multiple subpopulations of DNA, e.g. allelic variants, multiple mutant forms, and the like. Distinguishing the presence and identity of multiple sequences with current sequencing technology is virtually impossible, without additional work to isolate and perhaps clone the separate species of DNA.
A major advance in sequencing technology could be made if an alternative approach was available for sequencing DNA that did not require high resolution electrophoretic separations of DNA fragments, that generated signals more amenable to analysis, and that provided a means for readily analyzing DNA from heterozygous genetic loci.
An objective of the invention is to provide such an alternative approach to presently available DNA sequencing technologies.
SUMMARY OF THE INVENTION
The invention provides a method of nucleic acid sequence analysis based on repeated cycles of duplex extension along a single stranded template. Preferably, such extension starts from a duplex formed between an initializing oligonucleotide and the template. The initializing oligonucleotide is extended in an initial extension cycle by ligating an oligonucleotide probe to its end to form an extended duplex. The extended duplex is then repeatedly extended by subsequent cycles of ligation. During each cycle, the identity of one or more nucleotides in the template is determined by a label on, or associated with, a successfully ligated oligonucleotide probe. Preferably, the oligonucleotide probe has a blocking moiety, e.g. a chain-terminating nucleotide, in a terminal position so that only a single extension of the extended duplex takes place in a single cycle. The duplex is further extended in subsequent cycles by removing the blocking moiety and regenerating an extendable terminus.
In one aspect of the invention, a plurality of different initializing oligonucleotides is provided for separate samples of the template. Each initializing oligonucleo
Gorthey LeeAnn
Lu Frank
Lynx Therapeutics, Inc.
Macevicz Stephen C.
Powers Vincent M.
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