Recombinational cloning using nucleic acids having recombination sites

a nucleic acid and recombination technology, applied in the field of recombinant dna technology, can solve the problems of high background, toxic genes, long fragments, etc., and achieve the effects of less labor, high specificity, speed and yield, and improved specificity

Inactive Publication Date: 2010-10-21
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0034]The present invention provides nucleic acids, vectors and methods for obtaining amplified, chimeric or recombinant nucleic acid molecules using recombination proteins and at least one recombination site, in vitro or in vivo. These methods are highly specific, rapid, and less labor intensive than standard cloning or subcloning techniques. The improved specificity, speed and yields of the present invention facilitates DNA or RNA cloning or subcloning, regulation or exchange useful for any related purpose.

Problems solved by technology

A great deal of time and effort is expended both in the transfer of DNA segments from the initial cloning vectors to the more specialized vectors.
However, many other subclonings can take several weeks, especially those involving unknown sequences, long fragments, toxic genes, unsuitable placement of restriction sites, high backgrounds, impure enzymes, etc.
Accordingly, traditional subcloning methods, using restriction enzymes and ligase, are time consuming and relatively unreliable.
Although site specific recombinases have been used to recombine DNA in vivo, the successful use of such enzymes in vitro was expected to suffer from several problems.
Multiple DNA recombination products were expected in the biological host used, resulting in unsatisfactory reliability, specificity or efficiency of subcloning.
Thus, in vitro recombination reactions were not expected to be sufficiently efficient to yield the desired levels of product.

Method used

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  • Recombinational cloning using nucleic acids having recombination sites
  • Recombinational cloning using nucleic acids having recombination sites
  • Recombinational cloning using nucleic acids having recombination sites

Examples

Experimental program
Comparison scheme
Effect test

example 1

Recombinational Cloning Using Cre and Cre & Int

[0252]Two pairs of plasmids were constructed to do the in vitro recombinational cloning method in two different ways. One pair, pEZC705 and pEZC726 (FIG. 2A), was constructed with loxP and att sites, to be used with Cre and λ integrase. The other pair, pEZC602 and pEZC629 (FIG. 3A), contained the loxP (wild type) site for Cre, and a second mutant lox site, loxP 511, which differs from loxP in one base (out of 34 total). The minimum requirement for recombinational cloning of the present invention is two recombination sites in each plasmid, in general X and Y, and X′ and Y′. Recombinational cloning takes place if either or both types of site can recombine to form a Cointegrate (e.g. X and X′), and if either or both can recombine to excise the Product and Byproduct plasmids from the Cointegrate (e.g. Y and Y′). It is important that the recombination sites on the same plasmid do not recombine. It was found that the present recombinational c...

example 2

Using In Vitro Recombinational Cloning to Subclone the Chloramphenicol Acetyl Transferase Gene into a Vector for Expression in Eukaryotic Cells (FIG. 4A)

[0263]An Insert Donor plasmid, pEZC843, was constructed, comprising the chloramphenicol acetyl transferase gene of E. coli, cloned between loxP and attB sites such that the loxP site was positioned at the 5′-end of the gene (FIG. 4B). A Vector Donor plasmid, pEZC1003, was constructed, which contained the cytomegalovirus eukaryotic promoter apposed to a loxP site (FIG. 4C). One microliter aliquots of each supercoiled plasmid (about 50 ng crude miniprep DNA) were combined in a ten microliter reaction containing equal parts of lambda integrase buffer (50 mM Tris-HCl, pH 7.8, 70 mM KCl, 5 mM spermidine, 0.5 mM EDTA, 0.25 mg / ml bovine serum albumin) and Cre recombinase buffer (50 mM Tris-HCl, pH 7.5, 33 mM NaCl, 5 mM spermidine, 0.5 mg / ml bovine serum albumin), two units of Cre recombinase, 16 ng integration host factor, and 32 ng lambda...

example 3

Subcloned DNA Segments Flanked by AttB Sites without Stop Codons

Part I: Background

[0264]The above examples are suitable for transcriptional fusions, in which transcription crosses recombination sites. However, both attR and loxP sites contain multiple stop codons on both strands, so translational fusions can be difficult, where the coding sequence must cross the recombination sites, (only one reading frame is available on each strand of loxP sites) or impossible (in attR or attL).

[0265]A principal reason for subcloning is to fuse protein domains. For example, fusion of the glutathione S-transferase (GST) domain to a protein of interest allows the fusion protein to be purified by affinity chromatography on glutathione agarose (Pharmacia, Inc., 1995 catalog). If the protein of interest is fused to runs of consecutive histidines (for example His6), the fusion protein can be purified by affinity chromatography on chelating resins containing metal ions (Qiagen, Inc.). It is often desirab...

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Abstract

Recombinational cloning is provided by the use of nucleic acids, vectors and methods, in vitro and in vivo, for moving or exchanging segments of DNA molecules using engineered recombination sites and recombination proteins to provide chimeric DNA molecules that have the desired characteristic(s) and/or DNA segment(s).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The present application is a continuation of U.S. Application No. 09 / 648,790, filed Aug. 28, 2000, which is a continuation of U.S. application Ser. No. 09 / 177,387, filed Oct. 23, 1998, which claims the benefit of the filing date of U.S. Provisional Application No. 60 / 065,930, filed Oct. 24, 1997. The present application is also a continuation-in-part of U.S. application Ser. No. 09 / 432,085, filed Nov. 2, 1999, which is a divisional of U.S. application Ser. No. 09 / 233,493, filed Jan. 20, 1999 (now U.S. Pat. No. 6,143,557), which is a continuation of U.S. application Ser. No. 08 / 663,002, filed Jun. 7, 1996 (now U.S. Pat. No. 5,888,732), which is a continuation-in-part of U.S. application Ser. No. 08 / 486,139, filed Jun. 7, 1995 (now abandoned). The disclosures of which applications are incorporated by reference herein in their entireties.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates to recombin...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N1/21C12N15/74A61K48/00C12N15/09C07K19/00C12N1/15C12N1/19C12N5/10C12N9/00C12N15/10C12N15/64C12N15/66
CPCC12N9/00C12N15/66C12N15/64C12N15/10
Inventor HARTLEY, JAMES L.BRASCH, MICHAEL A.TEMPLE, GARY F.FOX, DONNA K.
Owner LIFE TECH CORP
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