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Compositions and methods for recombinational cloning of nucleic acid molecules

a nucleic acid and recombinant technology, applied in the field of recombinant dna technology, can solve the problems of toxic genes, long fragments, toxic dna, etc., and achieve the effects of improving specificity, speed and yield, and reducing labor intensity

Inactive Publication Date: 2008-10-30
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides methods and compositions for cloning and subcloning nucleic acid molecules using recombinational cloning. These methods are specific, rapid, and less labor-intensive than standard cloning techniques. The invention involves combining an insert donor molecule with a vector donor molecule and a recombination protein, and optionally a ribosomal protein. The resulting combination is then incubated under conditions sufficient to transfer the desired nucleic acid segments into the vector donor molecule. The invention also relates to methods of cloning or subcloning one or more desired nucleic acid molecules by recombinational cloning using different combinations of insert donor molecules, vector donor molecules, and recombination proteins. The methods and compositions of the invention can be used for various purposes, such as research, development, and medical applications.

Problems solved by technology

A great deal of time and effort is expended 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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  • Compositions and methods for recombinational cloning of nucleic acid molecules
  • Compositions and methods for recombinational cloning of nucleic acid molecules
  • Compositions and methods for recombinational cloning of nucleic acid molecules

Examples

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example 1

Stimulation of Integrase by E. coli Ribosomal Proteins

Materials and Methods

DNAs for Recombination Assays

[0230]Plasmid pHN894 (FIG. 2), bearing an attP site, and plasmid pBB105 (FIG. 3), bearing an attB site, are described (Kitts, P. A. and Nash, H. A. J. Mol. Biol. 204: 95-107 (1988); Nash, H. A. Methods Enz. 100: 210-216 (1983)). pBB105 was cut with EcoRI before use. Plasmid pHN872 (FIG. 4), bearing an attL site, and plasmid pHN868 (FIG. 5), bearing an attR site, are described (Kitts, P. A. and Nash, H. A. J. Mol. Biol. 204:95-107 (1988)). pHN872 was cut with SalI before use. These plasmids were propagated in E. coli strain DH10B. To grow cells for preparation of plasmid DNA, the growth medium contained in one liter: 12 g of tryptone, 24 g of yeast extract, 2.3 g of KH2PO4, 12.5 g of K2HPO4, 0.01% (v / v) PPG antifoam, and appropriate antibiotic. Cells from a glycerol seed were placed in 25 ml of medium containing 100 μg / ml ampicillin (pBB105, pHN894, pHN868) or 100 μg / ml kanamycin (...

example 2

Stimulation of Integrase Recombination by other 1, E. coli Ribosomal Proteins

[0286]In addition to S20 and L27, other E. coli ribosomal proteins may stimulate the activity of recombination systems, particularly the 1 Int system. In particular, E. coli ribosomal proteins that are basic and are about 14 kilodaltons or less in size are used to stimulate the activity of prokaryotic recombination systems. Such ribosomal proteins that may be used are shown in Table 5:

TABLE 5Additional Ribosomal Proteins for Use in Stimulating RecombinationActivityRibosomalNo. of Basic ResiduesNo. of TotalMolecular WeightProtein(% of Total)Residues(Daltons)S1017 (16.5%)10311,736S1423 (23.7%)9711,063S1516 (18.4%)8710,001S1614 (17.1%)829,191S1716 (19.3%)839,573S1817 (23.0%)748,896S1919 (20.9%)9110,299S2123 (32.9%)708,369L2117 (16.5%)10311,565L2321 (21.2%)9911,013L2422 (21.4%)10311,185L2517 (18.1%)9410,694L2818 (23.4%)778,875L2912 (19.0%)637,274L3010 (17.2%)586,411L3112 (19.4%)626,971L3211 (19.6%)566,315L3315 ...

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Abstract

The present invention relates generally to compositions and methods for enhancing recombinational cloning of nucleic acid molecules. In particular, the invention relates to compositions comprising one or more ribosomal proteins and one or more additional protein components required for recombinational cloning. More particularly, the invention relates to such compositions wherein the ribosomal proteins are one or more E. coli ribosomal proteins, still more particularly wherein the ribosomal proteins are selected from the group of E. coli ribosomal proteins consisting of S10, S14, S15, S16, S17, S18, S19, S20, S21, L20, L21, and L23 through L34, and most particularly S20, L27, and S15. The invention also relates to the use of these compositions in methods for recombinational cloning of nucleic acids, in vitro and in vivo, to provide chimeric DNA molecules that have particular characteristics and / or DNA segments. The invention also relates to isolated nucleic acid molecules produced by the methods of the invention, to vectors comprising such nucleic acid molecules, and to host cells comprising such nucleic acid molecules and vectors.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This is a divisional of U.S. application Ser. No. 10 / 292,838, filed Nov. 13, 2002, which is a divisional of U.S. application Ser. No. 09 / 438,358, filed Nov. 12, 1999, now U.S. Pat. No. 6,964,861, which claims the benefit of U.S. Provisional Application No. 60 / 108,324 filed Nov. 13, 1998, the contents of which are incorporated by reference herein in their entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates generally to recombinant DNA technology. The invention relates more specifically to compositions and methods for recombinational cloning of nucleic acid molecules using recombination systems. In particular, the invention relates to compositions comprising one or more ribosomal proteins, preferably one or more prokaryotic ribosomal proteins and particularly one or more E. coli ribosomal proteins, and one or more additional components required for recombinational cloning (such as one or mo...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/12C12N15/00C12N9/00C07K14/245C12N15/10C12N15/66
CPCC07K14/245C12N15/10C12N15/66
Inventor GERARD, GARY F.FLYNN, ELIZABETHHU, A-LI W.
Owner LIFE TECH CORP
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