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Column for DNA separation by matched ion polynucleotide chromatography
| Details |
Inventors: Gjerde, Douglas T.; Haefele, Robert M.; Hecker, Karl H.; Roque, Raquel R.;
Assignee: Transgenomic, Inc. (San Jose, CA)
Primary Examiner: Therkorn; Ernest G.
Assistant Examiner:
Attorney, Agent or Firm: Walker; William B.
An improved separation column and method for separating a mixture of double stranded DNA fragments by Matched Ion Polynucleotide Chromatography. The cylindrical column has an ID greater than about 5 mm and contains polymer beads. The beads have an average diameter of 1 to 100 microns and are unsubstituted polymer beads or are polymer beads substituted with a hydrocarbon moiety having from 1 to 1,000,000 carbons. The preferred beads are characterized by being substantially free from multivalent cations which are free to bind with DNA. The improved column provides enhanced separation of DNA fragments with sizes ranging from about 100 to 20,000 base pairs. The column also provides enhanced separation of heteroduplex and homoduplex DNA molecules in a mutation detection procedure in which the chromatography is performed under conditions effecting partial denaturation of DNA at a site of mismatched base pairs. |
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DETAILED DESCRIPTION It is an object of the present invention to provide a MIPC separation column which gives improved separation of polynucleotides; which allows separation of polynucleotides having lengths greater than about 1000; and which gives improved separation of homoduplex and heteroduplex DNA under partially denaturing conditions.
In one aspect, the invention concerns an improved separation column for separating a mixture of double stranded DNA fragments by Matched Ion Polynucleotide Chromatography (MIPC).
The mixture contains fragments having lengths exceeding about 1000 base pairs.
The column includes a cylinder having an ID greater than about 5 mm and containing polymer beads.
The beads have an average diameter of 1 to 100 microns and are unsubstituted polymer beads or are polymer beads substituted with a hydrocarbon moiety having from 1 to 1,000,000 carbons.
The preferred beads are characterized by being substantially free from multivalent cations which are free to bind with DNA.
In one embodiment, the column ID is greater than about 7 mm.
In another embodiment, the column ID is greater than about 10 mm.
In yet another embodiment, the column ID is greater than about 50 mm.
In still another embodiment the column ID is in the range of about 5 mm to about 1 m.
In another aspect, the invention concerns an improved method for separating a mixture of double stranded DNA fragments by MIPC in which the mixture includes fragments having lengths exceeding about 1000 base pairs.
The method includes a first step (a) of applying a solution of the DNA fragments fragments and counterion reagent to separation beads.
The beads are retained within a separation column.
The column has an ID greater than about 5 mm.
The beads have an average diameter of 1 to 100 microns, and are composed of unsubstituted polymer beads or polymer beads substituted with a hydrocarbon moiety having from 1 to 1,000,000 carbons.
The preferred beads are characterized by being substantially free from multivalent cations which are free to bind with DNA
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