High fidelity PCR cloning

Details High fidelity PCR cloning High fidelity PCR cloning

2001-02-14

2003-05-20

Fredman, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S091210, C536S024330

Reexamination Certificate

active

06566067

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a methodology for high fidelity cloning of target nucleic acids. The invention involves application of polymerase chain reaction (PCR) for a few cycles, which minimizes number of doublings of target sequences, and hence greatly reduces generation of polymerase-induced mutant fraction in PCR products. The invention also describes cloning of such high fidelity PCR products in a positive selection vector.
BACKGROUND OF THE INVENTION
Polymerase chain reaction or PCR (Saiki et al., 1985, Science 230, 1350-1354; Mullis and Faloona, 1987, Method Enzymol. 155, 335-350; U.S. Pat. Nos. 4,683,195; 4,683,202 and 4,965,188) has revolutionized amplification of target nucleic acids. The technique involves repeated cycles of denaturation of template nucleic acid molecules, sequence-specific primer annealing and primer extension using DNA polymerase thus resulting in an exponential amplification of the target nucleic acids. Usually, 30 cycles of PCR result in one million-fold amplification of target sequences from 20 doublings.
The PCR product itself could be used for diagnosis, quantitation of the template, direct sequencing and some other applications (U.S. Pat. Nos. 5,856,144; 5,487,993 and 5,891,687). However, for applications such as mutation analysis, identification of polymorphic transcripts, making RNA probes, sequencing, gene expression etc., usually a large quantity of DNA is needed. Thus it is necessary to isolate a bacterial clone carrying the PCR generated target DNA fragment in a cloning vector.
For correct structural and functional analyses it is fundamentally important to clone wild-type nucleic acids. Cloning of wild-type nucleic acids is critical for mutation analysis, identification of polymorphic transcripts and sequencing. It is also of paramount importance that for accurate functional analysis a true copy of the desired gene should be cloned in a suitable expression vector. However, an inherent disadvantage of DNA amplification using PCR is the introduction of mutations by the polymerases during synthesis of new DNA (Kunkel and Bebenek, 2000, Annu. Rev. Biochem. 69, 497-529; Andre et al., 1997, Genome Res. 7, 843-852; Cline et al., 1996, Nucl. Acids Res. 24, 3546-3551; Kong et al., 1993, J. Biol. Chem. 268, 1965-1975; Cariello et al., 1991, Nucl. Acids Res. 19, 4193-4198; Lundberg et al., 1991, Gene 180, 1-6; Reiss et al., 1990, Nucl. Acids Res. 18, 973-978; Keohavong and Thilly, 1989, Proc. Natl. Acad. Sci. USA 86, 9253-9257; Keohavong et al., 1988, DNA 7, 63-70; Kunkel et al., 1984, J. Bio. Chem. 259, 1539-1545; Loeb and Kunkel, 1982, Annu. Rev. Biochem. 52, 429-457). The total mutant fraction in a newly synthesized PCR-amplified DNA pool depends on the length of target sequence, error rate of a DNA polymerase, and number of doublings of the target sequence (Kunkel and Bebenek, 2000, Annu. Rev. Biochem. 69, 497-529; Andre et al., 1997, Genome Res. 7, 843-852; Cariello et al., 1991, Nucl. Acids Res. 19, 4193-4198; Reiss et al., 1990, Nucl. Acids Res. 18, 973-978). The error rates of different DNA polymerases vary from 1.3×10
−4
to 6.5×10
−7
mutant per basepair per doubling (Andre et al., 1997, Genome Res. 7, 843-852; Cline et al., 1996, Nucl. Acids Res. 24, 3546-3551; Cariello et al., 1991, Nucl. Acids Res. 19, 4193-4198; Keohavong and Thilly, 1989, Proc. Natl. Acad. Sci. USA 86, 9253-9257). For a million-fold amplification of target sequences 20 doublings are required, which are usually achieved by 30 cycles of PCR amplification. For a specific target sequence the increase of mutation fraction in the PCR-amplified DNA pool is a linear function of the number of doublings of the target sequence provided that the error rate of DNA polymerase remains constant under the specific PCR conditions (Kunkel and Bebenek, 2000, Annu. Rev. Biochem. 69,497-529; Andre et al., 1997, Genome Res. 7, 843-852; Cariello et al., 1991, Nucl. Acids Res. 19, 4193-4198; Reiss et al., 1990, Nucl. Acids Res. 18, 973-978).
The present methods of PCR cloning involve usually cloning of PCR products obtained after 20 doublings of target sequences thus resulting in generation of significant number of mutant clones, especially in case of cloning large target DNA fragments. Consequently, sometime rigorous sequencing of many clones is required to isolate a correct clone, and very often site-directed mutagenesis is necessary to correct a mutant clone. Furthermore, failure of PCR cloning of a correct sequence necessitates laborious screening of genomic or cDNA libraries, which usually represent correct sequences.
OBJECTS OF THE INVENTION
The primary object of this invention is to develop a methodology for cloning of high fidelity PCR products that contain no or minimum mutations. The invention aims to generate high fidelity PCR products by amplifying target sequences only for a few cycles, which minimizes number of doublings of target sequences, and hence greatly reduces polymerase-induced mutant fraction in PCR products. The invention further aims to clone high fidelity PCR products in a suitable vector.
Elimination of disadvantages associated with present protocols of PCR cloning and library screening is greatly desirable. The present invention aims to achieve a milestone advancement in cloning of a target sequence with no mutation.
SUMMARY OF THE INVENTION
The present invention describes a methodology of high fidelity PCR cloning of target nucleic acids. During PCR amplification of target nucleic acid sequences the polymerase-induced mutation fraction is linearly proportional to the number of doublings of the target sequences. The invention uses PCR on target nucleic acid sequences only for a few cycles, which minimizes number of doublings of target sequences, and hence greatly reduces polymerase-induced mutant fraction in PCR products. The high fidelity PCR products thus obtained are then cloned into a suitable vector. As an example, a 960 bp target sequence from
E. coli
DNA was amplified using PCR only for 3 cycles, and it was then directly cloned into a positive selection cloning vector pRGR2Ap. All insert-carrying clones showed cloning of functionally wild-type target DNA sequences, which indicated that the cloned target sequences most probably contained no mutation. Cloning of PCR products obtained from 3 cycles of amplification, instead of 30 cycles of amplification, theoretically achieves 10-fold reduction of mutations in the cloned fragments. The invention also envisions cloning of high fidelity products of primer extension.
BRIEF DESCRIPTION OF FIGURES
Not Applicable
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to develop a PCR cloning method that will greatly reduce, if not eliminate, the number of mutant clones that are observed with present PCR cloning experiments. Presently, in a typical PCR cloning experiment PCR products obtained usually from 30 cycles of PCR amplification are used in cloning. PCR amplification of target sequences for 30 cycles usually result in one million-fold amplification of target sequences, which is equivalent to 20 doublings of target sequences. The increase in total mutant fraction in PCR products pool is a linear function of the number of doublings of target sequences (Kunkel and Bebenek, 2000, Annu. Rev. Biochem. 69, 497-529; Andre et al., 1997, Genome Res. 7, 843-852; Cariello et al., 1991, Nucl. Acids Res. 19, 4193-4198; Reiss et al., 1990, Nucl. Acids Res. 18, 973-978). The invention describes generation of high fidelity PCR products by amplifying the target sequences only for a few cycles, which minimizes number of doublings of target sequences, and hence greatly reduces polymerase-induced mutant fraction in PCR products. The invention further describes cloning of such high fidelity PCR products in a suitable vector.
The amount of DNA obtained after a few cycles of PCR amplification is very small, and hence cloning of such small amount of DNA should give only a few colonies even at the most efficient conditions of ligation and transformation. Thus it

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