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Method for determination of gene function

a gene function and gene technology, applied in the field of molecular biology and microbiology, can solve the problems of limited knowledge of restriction sites, time-consuming and laborious, and relatively few htp methods to da

Inactive Publication Date: 2005-03-10
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0053] Genetic markers used in the present method may be selectable or screenable and may incorporate genes useful for the construction of

Problems solved by technology

However, relatively few HTP methods have been developed to date.
The above methods have worked well for disruption of genes in a range of organisms but have several inherent limitations, including being limited to knowledge of restriction sites, the unpredictable effects of co-suppression or gene silencing as well as being time consuming and labor intensive.
However, to date the use of In vitro transposition for making chromosomal mutations has been limited to the naturally transformable microorganisms (e.g., Haemophilus influenzae).
Because the above two systems use linear DNA in the transformations , single-crossover events cannot be obtained.
Thus, the above systems are not useful to making mutations in essential genes that are involved in cell survival.
Another limitation is that the above systems cannot be used to determine the functions of unknown genes on a genomic scale.

Method used

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  • Method for determination of gene function
  • Method for determination of gene function
  • Method for determination of gene function

Examples

Experimental program
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Effect test

example 1

[0112] Construction Of Genomic Library In Chromosomal Intepration Vector Isolation Of Chromosomal DNA

[0113] A “seed” culture of E. coli strain W3110 was prepared by inoculating an isolated colony into 5 mL of LB broth. The culture was grown overnight with shaking at 37° C. The following day a 1:1000 dilution of the seed culture was made into fresh LB broth and the culture was grown to an OD600 equal to 0.7-0.8. The cells were pelleted and resuspended into 2.5 mL of 10 mM Tris-EDTA. A hundred microliter of lysozyme (5 mg / mL) was added to the cells. Following a 15-minute incubation at 37° C., 0.1- mL of proteinase K and 0.125 mL of sodium doceyl sulfate was added to the cell suspension. The cell lysate was incubated for an additional 50-minutes at 37° C. The cell lysate was extracted twice with equal volumes of phenol, twice with a 1:1 ratio of phenol / chloroform, and twice with equals of chloroform. {fraction (1 / 10)} volume of a 3M sodium acetate solution and 2× volume of 100% ethano...

example 2

In vitro Transiposition

[0116] In vitro Transposition Reactions Using A Tn5-Based Transposition System

[0117] The E. coli W3110 DNA fragments were randomly mutagenized using the EZ::TN™ Insertion Kit (Epicentre Technologies, Madison, Wis.). The transposon donor DNA used in the transposition reactions was the EZ::TN™ Transposon (a linear DNA fragment), which carries the gene that confers resistance to Kanamycin between the ends of the transposable element. Per Epicentre's instructions, 0.2 μg of the target DNA (E. coli W3110 DNA fragments) was incubated with molar equivalents of the EZ::TN™ Transposon and 1U of the EZ::TN™ transposase. The reaction mixture was incubated for 2 hours at 37° C. The transposition reaction was stopped by adding 1 μL of the 10× Stop Solution and incubating the mixture for 10 minutes at 70° C.

[0118] In vitro Transposition Reactions Using A Tn7-Based Transposon System

[0119] The E. coli W3110 DNA fragments were randomly mutagenized using the GPS™-1 Genome P...

example 3

DNA Library Construction

[0120] Preparation of the Tn5-Based Mutant DNA Library

[0121] Sau3A digested W3110 DNA fragments (50 ng) containing the EZ::TN (Tn5-based) insertions (Kanamycin-resistant) were ligated into the dephosphorylated BamHI of pTSCSE7 (50 ng). The plasmid pTSCSE7 is a derivative of temperature sensitive vector pTSC29 and contains a gene that confers resistance to Chloroamphenicol (G. J. Phillips Plasmid (1999) 41, 78-81). pTSCSE7 also contain the npr-sacB gene from pBE83, which confers sensitivity to sucrose (V. Nagarajan, H. Albertson, M. Chen and J. Ribbe Gene (1992) 114, 121-126). The ligation reactions were done according to manufacturing specifications (New England Biolabs, Beverly, Mass.) and were incubated overnight at 16° C.

[0122] Any temperature-sensitive replicon can be used in the preparation of the mutant DNA library. The vector of choice must contain a selectable marker that is different from selectable marker that is present between transposon ends....

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Abstract

The present method is useful for the identification of genes, ORF's and other nucleic acid molecules which are essential for the expression of a specific phenotype in microorganisms. The method employs in vitro transposition in conjunction with an chromosomal integration vector containing a specific gene or genetic element whose function is unknown. Subsequent transformation of a recombination proficient host with the vector and growth first under non-integrating conditions and then under integrating conditions, followed by a selection screen for either single or double crossover events results in transformation that may be subjected to phenotypic screens to determine gene function.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 60 / 191,561, filed Mar. 23, 2000.FIELD OF THE INVENTION [0002] The invention relates to the field of molecular biology and microbiology. More specifically, a method has been developed whereby In vitro transposition is used to identify chromosomally integrated nucleic acid molecules having unknown function. BACKGROUND OF THE INVENTION [0003] With the advent of large-scale genome sequencing efforts, enormous amounts of sequence information is being made available to the research community on a daily basis. Genome sequencing efforts have been completed for the eukaryotes Saccharomyces cerevisiae and Caenorhabditis elegans and for several prokaryotes including Esherichia coli, Mycoplasma gentalium, Bacillus subtilus, Thermotoga maritima, Methanococcus jannaschii, including those of pharmaceutical interest. In spite of the volume of sequence data now available, only a small portion of the genes sequenced today ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68C12N15/09C12Q1/6897
CPCC12N15/1079C12Q1/6897C12N15/1082
Inventor SHARPE, PAMELANAGARAJAN, VASANTHA
Owner EI DU PONT DE NEMOURS & CO
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