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Solid phase technique for selectively isolating nucleic acids

a solid phase technique and nucleic acid technology, applied in the direction of nucleic acid reduction, microorganisms, biochemical apparatus and processes, etc., can solve the problem of limited throughput, achieve high ionic strength, improve the purity of exogenous dna of microparticles, and facilitate automation

Inactive Publication Date: 2006-01-05
WHITEHEAD INST FOR BIOMEDICAL RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013] The purity of the microparticle-bound exogenous DNA can be improved by washing the particle-bound nucleic acid molecules to remove other host cell biomolecules by contacting the microparticles with a high ionic strength wash buffer which dissolves, for example impurities (e.g., proteins, reagents or chemicals) adsorbed to the paramagnetic microparticles, but does not solubilize the adsorbed DNA. As a result, the exogenous DNA targeted for isolation remains adsorbed to the solid phase carrier surface. The washed, particle-bound exogenous DNA template can subsequently be removed from the solid phase carrier by contacting the washed microparticles with an elution buffer which solubilizes the adsorbed DNA, thereby preparing plasmid DNA suitable for use as a DNA nucleotide sequencing template.

Problems solved by technology

Although recent technological advancements and the advent of robotics have facilitated the automation of sequencing reactions and gel reading steps, throughput is still limited by the availability of readily automatable methods of nucleic acid purification.

Method used

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Examples

Experimental program
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Effect test

example 1

Isolation of Plasmid DNA Using PEG-Induced Separation and Paramagnetic Microparticles

[0063] This example provides a procedure, using the method described herein, to simultaneously process 96 individual plasmid miniprep samples from bacterial host cells comprising a pOT plasmid. The teaching of the instant disclosure provides ample guidance to allow an investigator of ordinary skill to modify this example to perform routing experimentation to derive a modification of this method that is capable of isolating exogenous DNA produced by the expression of alternative vectors (e.g., cosmids, BACs, P1s etc), in either high-copy- or low-copy-number, in numerous alternative host cells. Plasmid (e.g., exogenous) DNA was purified from the host cells by producing a mixture host cell lysate; preclearing the lysate of high molecular weight endogenous (e.g., host cell genomic) DNA by selectively precipitating it under conditions which promote its adsorption, but not the adsorption of pOT plasmid ...

example 2

PEG-Induced Size Selection of Sheared DNA for Shotgun Library Construction

[0082] Shortgun sequencing strategies enable the de novo determination of an unknown nucleotide sequence. The method imposes no limitation on the size of the starting DNA molecule whose sequence is to be determined and requires no prior knowledge of the nucleotide sequence of the DNA fragments selected as inserts for cloning. According to protocols which are well-known to those of skill in the art, the starting DNA molecule, whose sequence is to elucidated, s fragmented, either by enzymatic digestion or by physical shearing (e.g., using a nebulizer, sonicator or hydroshearing) to produce a shattered DNA library typically comprising 0.5-5 kb fragments. The shotgun strategy then requires that a subfraction of these fragments characterized by a narrow size range (e.g., 0.5-1.0 kb, 0.8-1.5 kb, 1.0-1.5 kb) be selected for use as inserts into an appropriate DNA sequencing vector. The nucleotide sequences of the re...

example 3

Use of PEG-Induced Size Selection to Prepare Post Nucleotide Sequencing Reaction Extension Products for Capillary Electrophoresis

[0093] A conventional sequencing reaction comprises a mixture of a DNA template, numerous extension products, excess terminators or primers and nucleotides (e.g., both deoxy- and dideoxynucleotides) admixed with reagents (e.g., salts or alcohols). It is well known that the quality of the DNA sequence data, as assessed by average read length and unedited accuracy, is a direct correlate of the purity of the extension products used for electrophoretic analysis. Purity is an important factor for all sequencing methods, and is particularly crucial to the success of automated dye-labeled dideoxynucleotide sequencing methods. The following method has been used to prepare extension products (e.g., Sanger sequencing products) for capillary electrophoresis: [0094] Transfer a 20 ul aliquot of detemplated fluorescently labeled dye terminator or dye primer sequencing...

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Abstract

A method of isolating target nucleic acid molecules from a solution comprising a mixture of different size nucleic acid molecules, in the presence or absence of other biomolecules, by selectively facilitating the adsorption of a particular species of nucleic acid molecule to the functional group-coated surface of magnetically responsive paramagnetic microparticles is disclosed. Separation is accomplished by manipulating the ionic strength and polyalkylene glycol concentration of the solution to selectively precipitate, and reversibly adsorb, the target species of nucleic acid molecule, characterized by a particular molecular size, to paramagnetic microparticles, the surfaces of which act as a bioaffinity adsorbent for the nucleic acids. The target nucleic acid is isolated from the starting mixture based on molecular size and through the removal of magnetic beads to which the target nucleic acid molecules have been adsorbed. The disclosed method provides a simple, robust and readily automatable means of nucleic acid isolation and purification which produces high quality nucleic acid molecules suitable for: capillary electrophoresis, nucleotide sequencing, reverse transcription cloning the transfection, transduction or microinjection of mammalian cells, gene therapy protocols, the in vitro synthesis of RNA probes, cDNA library construction and PCR amplification.

Description

RELATED APPLICATION(S) [0001] This application is a divisional of U.S. application Ser. No. 10 / 346,714, filed Jan. 16, 2003, which is a continuation of U.S. application Ser. No. 09 / 311,317, filed May 13, 1999, which claims the benefit of U.S. Provisional Application No. 60 / 085,480, filed May 14, 1998 and U.S. Provisional Application No. 60 / 121,779, filed on Feb. 26, 1999. The entire teachings of the referenced applications are incorporated herein by reference.GOVERNMENT SUPPORT [0002] The invention was supported, in whole or in part, by a National Human Genome Research Institute Grant, Grant Number 5P 50 HG00098-09, from the National Institutes of Health. The United States Government has certain rights in the invention.BACKGROUND OF THE INVENTION [0003] Many molecular biology applications, such as capillary electrophoresis, nucleotide sequencing, reverse transcription cloning and gene therapy protocols, which contemplate the transfection, transduction or microinjection of mammalian ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12N1/08C12N15/10
CPCC12N15/1006C12N15/1013C12Q1/6806C12Q2563/143C12Q2527/137C12Q2523/308
Inventor MCKERNAN, KEVINMCEWAN, PAULMORRIS, WILLIAM
Owner WHITEHEAD INST FOR BIOMEDICAL RES
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