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Method for making insertional mutations

A technique of insertion mutation and synapse, applied in other methods of inserting foreign genetic materials, preparation of mutants, botanical equipment and methods, etc., can solve problems such as unsatisfactory and complicated analysis of insertion mutation library

Inactive Publication Date: 2001-10-24
WISCONSIN ALUMNI RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This unwanted redundant transposition is undesirable and complicates the analysis of insertional mutant libraries

Method used

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  • Method for making insertional mutations
  • Method for making insertional mutations
  • Method for making insertional mutations

Examples

Experimental program
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Embodiment

[0035] In a 1 μl, 0.05 μg purified hyperactive transposase (EK45 / MA56 / LP372) reaction, the amino acid sequence disclosed in the international application PCT / US97 / 15941 cited herein was combined with 0.1 μg of transposable polynucleotide, the transposable polynucleotide The locus polynucleotide contains an expression cassette encoding a protein that confers kanamycin resistance to target cells. This expression cassette is flanked by spliced ​​ends (as described in the same international application cited), and in image 3 displayed in . The polynucleotide is provided in a separate reaction, either as a supercoiled plasmid, as a linearized plasmid, or as a polynucleotide fragment including at its terminus the reverse sequence required for Tn5 transposase-mediated transposition.

[0036] With or without magnesium ions (Mg ++ ) in reaction buffer for 1 hour. In the absence of magnesium ions, synaptoplexes formed but did not transpose in vitro.

[0037] After incubation, 40 µl ...

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Abstract

A method for making insertional mutations at random or quasi-random locations in the chromosomal or extra-chromosomal nucleic acid of a target cell includes the step of combining, in the target cell, cellular nucleic acid with a synaptic complex that comprises (a) a Tn5 transposase protein and (b) a polynucleotide that comprises a pair of nucleotide sequences adapted for operably interacting with Tn5 transposase and a transposable nucleotide sequence therebetween, under conditions that mediate transpositions into the cellular DNA. In the method, the synaptic complex is formed in vitro under conditions that disfavor or prevent the synaptic complexes from undergoing productive transposition.

Description

[0001] There are no applicable cross-referenced related applications. [0002] No applicable jointly proposed research or development literature. Background of the invention [0003] Efficient insertion of exogenous nucleic acids into chromosomal and extrachromosomal nucleic acids of cells is a desired technique in the field of molecular biology to identify chromosomal regions involved in expressing peptides and proteins or regulating their expression. It can also be used advantageously in the development of novel therapeutics and pharmaceuticals as well. [0004] A commonly used method relies on in vivo Tn5 mutagenesis to insert a polynucleotide of interest into cellular DNA and construct a library of cells containing the inserted polynucleotide at random or quasi-random positions. The in vivo Tn5 mutagenesis approach requires the target cell to encode a transposase, whether native or produced from an introduced expression construct. In addition, it may be necessary to cons...

Claims

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Application Information

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IPC IPC(8): G01N33/50C12N9/00C12N9/22C12N15/00C12N15/01C12N15/09C12N15/10C12N15/90C12Q1/02G01N33/15G01N33/566
CPCC12N15/102C12N15/90C12N9/22
Inventor W·S·列兹尼科夫I·Y·戈雷申
Owner WISCONSIN ALUMNI RES FOUND
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