Method of error reduction in nucleic acid populations

• US 20040132029A1
• Filed: 02/28/2003
• Published: 07/08/2004
• Est. Priority Date: 02/28/2002
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Cerrina, Francesco, Belshaw, Peter J., Sussman, Michael R., Richmond, Kathryn, Kaysen, James H.
• Granted Patent: US 7,303,872 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase

Abstract

A method is disclosed for the direct synthesis of double stranded DNA molecules of a variety of sizes and with any desired sequence. The DNA molecule to be synthesis is logically broken up into smaller overlapping DNA segments. A maskless microarray synthesizer is used to make a DNA microarray on a substrate in which each element or feature of the array is populated by DNA of a one of the overlapping DNA segments. The complement of each segment is also made in the microarray. The DNA segments are released from the substrate and held under conditions favoring hybridization of DNA, under which conditions the segments will hybridize to form duplexes. The duplexes are then separated using a DNA binding agent which binds to improperly formed DNA helixes to remove errors from the set of DNA molecules. The segments can then be hybridized to each other to assemble the larger target DNA sequence.

Claims

1. A method for separation of separation of DNA molecules of correct sequence away from DNA molecule of incorrect sequence, the method comprising the steps of

(a) exposing a solution of double stranded DNA molecules to a DNA binding agent which will binds selectively to duplex DNA molecules having a sequence mismatch between its duplex strands;

and

(b) separating the DNA molecules to which the DNA binding agent bound from those DNA molecules to which the DNA binding agent did not bind.

2. A method as claimed in claim 1 wherein before the exposing step, the method includes the step of permitting single stranded DNA strands to hybridize to other single DNA strands.

3. A method as claimed in claim 1 wherein the separation is performed by affinity binding the DNA binding agent at a fixed location.

4. A method as claimed in claim 1 wherein the separation is performed by electrophoresis.

5. A method as claimed in claim 1 wherein the DNA binding agent is MutS.

6. A method as claimed in claim 1 wherein the steps (a) and (b) are performed repetitively until a desired level of purity of correct sequence is achieved.

7. A method for separating out duplex DNA molecules having a sequence error in a pool of duplex DNA molecules the majority of which are of a correct sequence, the method comprising the steps of

(a) denaturing the duplex DNA molecules;

(b) permitting the DNA molecules to hybridize to form new DNA duplex molecules;

(c) exposing the duplex DNA molecules to a DNA binding agent that binds selectively to DNA molecules having an irregularity in the topology of the DNA duplex; and

(d) separating the DNA molecules by separation out of those DNA molecules to which the DNA binding agent bound.

8. A method as claimed in claim 7 wherein the separation is performed by affinity binding the DNA binding agent at a fixed location.

9. A method as claimed in claim 7 wherein the separation is performed by electrophoresis.

10. A method as claimed in claim 7 wherein the DNA binding agent is MutS.

11. A method as claimed in claim 7 wherein steps (a) through (d) are repeated multiple times until a desired level of purity of DNA of correct sequence is obtained.

12. A method for making DNA sequences of defined sequence comprising the steps of

(a) making a microarray of single stranded DNA probes, the probes constructed so that each probe has a complementary portion that is partially complementary to another probe on the microarray and further constructed so that for each set of probes a complete complementary set of probes is constructed;

(b) releasing the single stranded DNA probes from the microarray;

(c) cooling the single stranded probes so that DNA duplexes are formed which are mainly formed of probes hybridized to their complete complementary probe;

(d) exposing the DNA duplexes to a DNA binding agent which will selectively bind to a DNA duplex which has an irregularity in its topographical shape;

(e) separating out the DNA duplexes to which the DNA binding agent bound;

(f) denaturing the DNA duplexes to released the single stranded DNA probes from the DNA duplexes;

(g) cooling the DNA duplexes under conditions which favor at least some of the single stranded DNA probes binding to the probes to which they are only partially complementary to form DNA complexes which are double stranded in at least some part; and

(h) extending the DNA complexes thus made to add a second DNA strand to remaining single stranded parts of the DNA complexes.

13. A method as claimed in claim 12 wherein the separation is performed by affinity binding the DNA binding agent at a fixed location.

14. A method as claimed in claim 12 wherein the DNA binding agent is MutS