Recombinant DNA processes using a dNTP mixture containing modified nucleotides

Abstract

Amplification mixtures, kits, amplicons, kits and processes are provided for amplifying a nucleic acid. In particular, provided are processes which utilize an amplification mixture comprising a polymerase, a deoxynucleotidetriphosphate (DNTP) mixture which contains modified dNTPs, a first primer and a second primer. Further provided are DNTP mixtures which contain modified dNTPs for at least two of the four nucleotide triphosphates, which when incorporated into a polynucelotide, impart resistance to enzymatic degradation by an exonuclease at the sites of incorporation of the modified dNTPs. Also provided are the amplicons and vectors which incorporate the modified nucleotides.

Description

[0001] This application is a division of U.S. patent application Ser. No. 10 / 002,292, filed Nov. 15, 2001, which claims priority to U.S. provisional patent application Ser. No. 60 / 325,612 filed Sep. 28, 2001, the entire contents of which are incorporated herein by reference.FIELD OF INVENTION [0002] The present invention relates to recombinant DNA processes using a deoxynucleotide triphosphate (DNTP) mixture containing modified dNTPs, which when incorporated into a polynucleotide, impart resistance against enzymatic degradation by an exonuclease at the sites of incorporation of the modified dNTPs. BACKGROUND OF THE INVENTION [0003] Many widely known recombinant DNA techniques involve replicating or amplifying DNA. One such example is the cloning of an insert DNA into a target DNA fragment. During this procedure, the target fragment is typically digested with a restriction enzyme such as EcoRI. Similarly, the insert DNA, having the gene of interest, is digested with the same enzyme. ...

AI Technical Summary

Benefits of technology

[0010] One aspect of the present invention, therefore, is the provision of materials, processes and kits for synthesizing DNA from a target nucleic acid. Generally, the present invention relates to processes which incorporate modified deoxynucleotide triphosphates (dNTPs) into a nucleic acid. The incorporation of these modified dNTPs impart resistance against enzymatic degradation by an exonuclease at the site of incorporation of the modified dNTPs thereby protecting the amplified product from complete degradation.

[0018] This dNTP mixture protects nucleotides from enzymatic degradation by an exonuclease thus increasing the efficiency and the ease of the cloning process. A further aspect of the present invention is to provide the dNTP mixture containing modified dNTPs for at least two of the four nucleotide triphosphates, which during the extension step of the PCR, are incorporated into the amplification product in lieu of one of the non-modified dNTPs.

Problems solved by technology

This method is limited to cloning DNA into EcoRI cleaved vectors because the EcoRI recognition site is the only commonly used restriction site whose 5′ four base overhang is punctuated by a base, i.e., guanine, not represented within the 5′ PAATTC overhang.

While the use of exonuclease III provides a relatively simple method for directional cloning, it may be difficult to control the length of the generated cohesive ends which may be critical when cloning small size insert DNA.

There are several drawbacks, however, with single restriction enzyme digestion cloning methods and double restriction enzyme digestion directional cloning methods.

For instance, digesting both the target DNA and insert DNA with restriction enzymes can be time consuming and multiple enzyme digestions increase the risk that either the target or insert DNA sequence will be cleaved at an internal restriction site.

Additionally, some restriction enzymes cleave very poorly, or not at all, when their recognition sequence is at or near the termini of a DNA strand.

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