The invention relates to the field of replicating, amplifying, and sequencing nucleic acids. Further, this invention relates to novel proteins that enhance the activity of the polymerases.
In vitro polymerization techniques have enormously benefited the fields of biotechnology and medicine. The ability to manipulate nucleic acids with polymerization reactions greatly facilitates techniques ranging from gene characterization and molecular cloning (including, but not limited to sequencing, mutagenesis, synthesis and amplification of DNA), determining allelic variations, and detecting and screening of various diseases and conditions (e.g., hepatitis B).
An in vitro polymerization technique of great interest is the polymerase chain reaction (PCR). This method rapidly and exponentially replicates and amplifies nucleic acids of interest. PCR is performed by repeated cycles of denaturing a DNA template, usually by high temperatures, then annealing opposing primers to the complementary DNA strands, and then extending the annealed primers with a DNA polymerase. Multiple cycles of PCR result in an exponential amplification of the DNA template.
Unfortunately, PCR has limitations. These limitations range from 1) the rate of nucleotide incorporation, 2) the fidelity of nucleotide incorporation, 3) the length of the molecule to be amplified, and 4) the specificity of the polymerase.
Various methods to improve PCR exist. One approach is to optimize the reaction conditions, e.g., such as the pH, dNTP concentrations, or reaction temperatures. Another approach is to add various chemical compounds, e.g., formamide (Sarkar, G., et al. Nucl. Acids Res. 18: 7465 (1990)), tetramethyammonium chloride, and dimethyl sulfoxide (Chevet et al., Nucl. Acids Res. 23:3343-3344 (1995); Hung et al., Nucl. Acids Res. 18:4953 (1990)) to either increase the specificity of the PCR reaction and/or increase yield. Other attempts include adding various proteins, such as replication accessory factors. Replication accessory factors known to be involved in DNA replication have also increased yields and the specificity of PCR products. For example, E. coli single-stranded DNA binding protein, such as RFA, has been used to increase the yield and specificity of primer extension reactions and PCR reactions (U.S. Pat. Nos. 5,449,603, and 5.534,407). Another protein, the gene 32 protein of phage T4, appears to improve the ability to amplify larger DNA fragments (Schwartz et al., Nucl. Acids Res. 18: 1079 (1990).
An important modification that has enhanced the ease and specificity of PCR is the use of Thermus aquaticus (Taq) DNA polymerase in place of the Klenow fragment of E. coli DNA pol I (Saiki et al., Science 230: 1350-1354 (1988)). The use of this thermostable DNA polymerase obviates the need for repeated enzyme additions, permits elevated annealing and primer extension temperatures, and enhances specificity. Further, this modification has enhanced the specificity of binding between the primer and its template. But, Taq polymerase has a fundamental drawback because it does not have 3′ to 5′ exonuclease activity and, therefore, cannot excise incorrect nucleotides added to the ends of the amplified products. Due to this limitation, the fidelity of Taq-PCR reactions typically have suffered. Therefore, those in the field have searched for another thermostable polymerase that has 3′ to 5′ exonuclease activity.
Polymerases having 3′ to 5′ exonuclease activity have been found in archaebacteria (archaea). Archaebacteria is a third kingdom, different from eukaryotes and bacteria (eubacteria). Many archaebacteria are thermophilic bacteria-like organisms that can grow in extremely high temperatures, i.e., 100° C. One such archaebacteria is Pyrococcus furiosus (Pfu). A monomeric polymerase from Pfu has been identified that has the desired 3′ to 5′ exonuclease activity and synthesizes nucleic acids of interest at high temperatures (Lundberg et al., Gene 108: 1-6 (1991); Cline et al., Nucl. Acids Res. 24: 3546-3551 (1996) (This polymerase is referred to as Pfu polymerase.)) A second DNA polymerase has been identified in P. furiosus which has two subunits (DP1/DP2) and is referred to as pol II. See References 1 and 15. This polymerase may also be enhanced by the accessory factors.
Certain natural proteins exist in archaebacteria, i.e., PEF (polymerase enhancing factors) that exhibit deoxyuracil triphosphatase (dUTPase) activity and that enhance the activity of Pfu polymerase (International Patent Application Publication No. WO 98/42860, published on Oct. 1, 1998). The presence of deoxyuracil-containing DNA in a DNA polymerization reaction inhibits polymerase activity (Lasken et al. (J. Biol. Chem. 271: 17692-17696)). Specifically, during the course of a normal PCR reaction, a dCTP may be deaminated into dUTP, thereby introducing a deoxyuridine into the newly synthesized DNA. But, when this newly synthesized DNA is thereafter amplified, the presence of the deoxyuridine inhibits the Pfu polymerase. The archaeal dUTPase (PEF) prevents dUTP incorporation and, thus, avoids the inhibition of the Pfu polymerase. Accordingly, the archaeal dUTPase optimizes the activity of Pfu polymerase.
According to certain embodiments, the invention provides methods of, and materials for, enhancing the polymerase activity of Pfu polymerase. Certain embodiments involve major components of the replication machinery in eukaryotes, e.g.: a helicase enzyme that unwinds the DNA helix and, thereby, provides a single-stranded DNA template; single-stranded DNA binding proteins (RFA) that bind and stabilize the resulting single-stranded DNA template; a “sliding clamp” protein (PCNA) that stabilizes the interaction between the polymerase and the primed single-stranded DNA template and that enhances synthesis of long DNA strands (also known as “processivity”); and a “clamp-loading” protein complex (RFC) that assembles the PCNA protein.
According to certain embodiments, the invention provides novel DNA replication accessory factors which have been isolated and purified from the hyperthermophilic archaeal bacteria Pyrococcus furiosus. In certain embodiments, the isolated proteins are thermostable homologues of eukaryotic DNA replication proteins PCNA, RF-C subunits, RFA, and helicases. Recent computer analysis of sequence data do not describe the proteins disclosed herein, although sequences that may be homologous to eukaryotic and/or eubacterial replication factors exist (Chedin et al., TIBS 23:273-277 (1998); Egdell and Doolittle, Cell 89: 995-998 (1997); Bult et al., Science 273: 1058-1-73 (1996)).
According to certain embodiments, this invention also involves isolated polynucleotides that encode the replication accessory factors.
In certain embodiments, the polynucleotide may be cDNA, genomic DNA, mRNA, or plasmid DNA.
According to certain embodiments, the invention includes vectors comprising a polynucleotide that encodes a replication accessory factor and host cells comprising such vectors. According to certain embodiments, the invention includes polypeptides expressed in those host cells. Further, this invention provides not only the host cells and their products, but also, the methods of using such host cells to produce the polypeptides of interest.
According to certain embodiments, the invention includes methods of enhancing a nucleic acid polymerase reaction comprising the addition of one or more of the replication accessory factors to the reaction.
In certain embodiments, only one archaeal replication accessory factor will be added into the nucleic acid polymerase reaction. In other embodiments, a combination of factors may be added.
In certain embodiments, an archaeal dUTPase may be combined with one or more of those replication accessory factors to further enhance the polymerase
In certain embodiments, this invention also provides methods of synthesizing nucleic acids comprising employing an archaeal polymerase and an archaeal replication accessory factor(s).
According to certain embodiments, the invention includes methods of amplifying nucleic acids of interest comprising employing an archaeal polymerase and an archaeal replication accessory factor(s).
In certain embodiments of the inventive methods, the archaeal polymerase is Pfu polymerase. In certain embodiments of those methods, the archaeal polymerase is combined with another polymerase, such as Taq. In other embodiments of the these methods, an archaeal dUTPase may also be included to enhance polymerase activity.
In certain embodiments of the inventive methods, the archaeal polymerase is P. furiosus pol II polymerase.
In certain embodiments, this invention also provides a kit used in the practice of the above-described methods.
In certain embodiments, this invention also provides a kit comprising an archaeal polymerase and at least one archaeal replication accessory factor.
In certain embodiments, those kits would also comprise an archaeal dUTPase and possibly, another polymerase, such as Taq.