Solid phase technique for selectively isolating nucleic acids

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 ...

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.

[0014] In one embodiment, the invention is a readily automatable method of isolating a plasmid DNA template for nucleotide sequencing. The use of paramagnetic microparticles having functional group-coated surfaces as a bioaffinity adsorbent for nucleic acid molecules affords an alternative, and readily automatable, means of molecular separation useful in the design of a solid phase technique for the selective isolation of nucleic acid molecules targeted for isolation. A particular advantage of the disclosed method is that it obviates the necessity of centrifugation and filtration steps and the use of organic solvents. In addition, the ample surface area provided by suitable solid phase carriers allows the volume of the reactions to be reduced, which not only facilitates automation, but which also eliminates the need for subsequent concentration steps which conventionally require the use of a solvent whose presence in the final preparation can adversely effect the quality of the isolated DNA. Thus, the invention offers purification methods which are simple, cost effective, robust and readily amenable to automation.

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.