Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-02-06
2001-10-23
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S174000, C530S350000, C530S382000, C530S388210
Reexamination Certificate
active
06306588
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the field of molecular and cellular biology. The invention relates to compositions and methods for use in analyzing and typing polymorphic regions of DNA. More particularly, the invention is directed to compositions of polymerases (preferably DNA polymerases and most preferably thermostable DNA polymerases), and methods using these compositions, whereby polymorphic, minisatellite, microsatellite or STR DNA fragments maybe amplified and analyzed. The compositions and methods of the present invention are useful in a variety of techniques employing DNA amplification and polymorphism analysis, including medical genetic, forensic, and plant breeding applications.
The present invention also relates to polymerases having reduced, substantially reduced or eliminated ability to add one or more non-templated nucleotides to the 3′ terminus of a synthesized nucleic acid molecule. Preferably, the polymerases of the invention are thermostable or mesophilic polymerases. Specifically, the polymerases of the present invention (e.g., DNA or RNA polymerases) have been mutated or modified to reduce, substantially reduce or eliminate such activity (compared to the unmodified, unmutated, or wild type polymerase), thereby providing a polymerase which synthesizes nucleic acid molecules having little or no non-templated 3′ terminal nucleotides. Such polymerases thus have enhanced or greater ability to produce a double stranded nucleic acid molecule having blunt ended termini which may facilitate cloning of such molecules. The present invention also relates to cloning and expression of the polymerases of the invention, to nucleic acid molecules containing the cloned genes, and to host cells which express said genes. The polymerases of the present invention may be used in DNA sequencing, amplification, nucleic acid synthesis, and polymorphism analysis.
The invention also relates to polymerases of the invention which have one or more additional mutations or modifications. Such mutations or modifications include those which (1) substantially reduce 3′→5′ exonuclease activity; and/or (2) substantially reduce 5′→3′ exonuclease activity. The polymerases of the invention can have one or more of these properties. These polymerases may also be used in nucleic acid analysis including but not limited to DNA sequencing, amplification, nucleic acid synthesis, and polymorphism analysis.
BACKGROUND OF THE INVENTION
DNA Structure
The genetic framework (i.e., the genome) of an organism is encoded in the double-stranded sequence of nucleotide bases in the deoxyribonucleic acid (DNA) which is contained in the somatic and germ cells of the organism. The genetic content of a particular segment of DNA, or gene, is only manifested upon production of the protein which the gene ultimately encodes. There are additional sequences in the genome that do not encode a protein (i.e., “noncoding” regions) which may serve a structural, regulatory, or unknown function. Thus, the genome of an organism or cell is the complete collection of protein-encoding genes together with intervening noncoding DNA sequences. Importantly, each somatic cell of a multicellular organism contains the full complement of genomic DNA of the organism, except in cases of focal infections or cancers, where one or more xenogeneic DNA sequences may be inserted into the genomic DNA of specific cells and not into other, non-infected, cells in the organism.
Minisatellite and Microsatellite DNA
Interspersed throughout the genomic DNA of most eukaryotic organisms are short stretches of polymorphic repetitive nucleotide sequences known as “minisatellite DNA” sequences or fragments (Jeffreys, A. J., et al,
Nature
314:67-73 (1985)). These repeating sequences often appear in tandem and in variable numbers within the genome, and they are thus sometimes referred to as “short tandem repeats” (“STRs”) or “variable numbers of tandem repeats” (“VNTRs”) (see U.S. Pat. No. 5,075,217; Nakamura et al.,
Science
235:1616-1622 (1987)). Typically, however, minisatellite repeat units are about 9 to 60 bases in length (Nakamura et al.,
Science
235:1616-1622 (1987); Weber and May,
Am. J. Hum. Genet
. 44:388-396 (1989)) which are repeated in tandem about 20-50 times (Watson, J. D., et al., eds.,
Recombinant DNA
, 2nd ed., New York: Scientific American Books, p. 146 (1992)). Other short, simple sequences which are analogous to minisatellite DNAs, termed “microsatellite DNAs” (Litt, M., and Luty, J. A.,
Am. J. Hum. Genet
44:397-401 (1989); Weber and May,
Am. J. Hum. Genet
. 44:388-396 (1989)), are usually about 1-6 bases in repeat unit length and thus give rise to monomeric (Economou, E. T., et al,
Proc. Natl. Acad Sci. USA
87:2951-2954 (1990)), dimeric, trimeric, quatrameric, pentameric or hexameric repeat units (Litt, M., and Luty, J. A.,
Am. J. Hum. Genet
44:397-401 (1989); Weber and May,
Am. J. Hum. Genet
. 44:388-396 (1989)). The most prevalent of these highly polymorphic microsatellite sequences in the human genome is the dinucleotide repeat (dC-dA)
n
•(dG-dT)
n
(where n is the number of repetitions in a given stretch of nucleotides), which is present in a copy number of about 50,000-100,000 (Tautz, D., and Renz, M.,
Nucl. Acids Res
. 12:4127-4138(1984); Dib, C., et al.,
Nature
360:152-154 (1996)), although the existence of a variety of analogous repeat sequences in the genomes of evolutionarily diverse eukaryotes has been reported (Hamada, H., et al.,
Proc. Natl. Acad Sci. USA
79:6465-6469 (1982)).
The actual in vivo function of minisatellite and microsatellite sequences is unknown. However, because these tandemly repeated sequences are dispersed throughout the genome of most eukaryotes, exhibit size polymorphism, and are often heterozygous (Weber, J. L.,
Genomics
7:524-530 (1990)), they have been explored as potential genetic markers in assays attempting to distinguish closely related individuals, and in forensic and paternity testing (see, e.g., U.S. Pat. No. 5,075,217; Jeffreys, A. J., et al.,
Nature
332:278-281 (1988)). The finding that mutations often are observed in microsatellite DNA regions in cancer cells (Loeb, L. A.,
Cancer Res
. 54:5059-5063 (1994)), potentially linking genomic instability to the carcinogenic process and providing useful genetic markers of cancer, lends additional significance to methods facilitating the rapid analysis and genotyping of polymorphisms in these genomic DNA regions.
Methods of Genotyping Minisatellite or STR DNA Sequences
To analyze minisatellite, microsatellite or STR DNA sequence polymorphisms, a variety of molecular biological techniques have been employed. These techniques include restriction fragment length polymorphism (RFLP) or “DNA fingerprinting” analysis (Wong, Z., et al.,
Nucl. Acids Res
. 14:4605-4616 (1986); Wong, Z., et al.,
Ann. Hum. Genet
51:269-288 (1987); Jeffreys, A. J., et al.,
Nature
332:278-281 (1988); U.S. Pat. Nos. 5,175,082; 5,413,908; 5,459,039; and 5,556,955). Far more commonly employed for STR genotyping than RFLP and hybridization, however, are amplification-based methods, such as those relying on the polymerase chain reaction (PCR) method invented by Mullis and colleagues (see U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159). These methods use “primer” sequences which are complementary to opposing regions flanking the polymorphic DNA sequence to be amplified from the sample of genomic DNA to be analyzed. These primers are added to the DNA target sample, along with excess deoxynucleotides and a DNA polymerase (e.g., Taq polymerase; see below), and the primers bind to their target via base-specific binding interactions (i.e., adenine binds to thymine, cytosine to guanine). By repeatedly passing the reaction mixture through cycles of increasing and decreasing temperatures (to allow dissociation of the two DNA strands on the target sequence, synthesis of complementary copies of each strand by the polymerase, and re-annealing of the new complementary strands), the copy number of the
Chatterjee Deb K.
Solus Joseph
Yang Shuwei
Invitrogen Corporation
Jones W. Gary
Sterne Kessler Goldstein & Fox P.L.L.C.
Tung Joyce
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