Method of error reduction in nucleic acid populations

• US 7,303,872 B2
• Filed: 02/28/2003
• Issued: 12/04/2007
• Est. Priority Date: 02/28/2002
• Status: Expired due to Fees

• 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.
• Primary Examiner(s): Chunduru; Suryaprabha
• Application Number:
  US10/376,720
• Publication Number:
  US 20040132029A1
• Time in Patent Office: 1,740 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.31, 536/24.32
• US Class Current: 435/6.16
• 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 making DNA sequences of pre-selected defined sequence using a microarray synthesis process that makes occasional errors, the method 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, a minority of the probes in the microarray being erroneously made with a sequence not the defined sequence;

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

(c) cooling the single stranded probes so that DNA duplexes are formed, the duplexes formed of probes hybridized to their complete complementary probe having the defined sequence while duplexes formed with single stranded DNA probes not of the defined sequence having an irregularity in its topographical shape;

(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 release 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.

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

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

4. A method for making DNA sequences of pre-selected defined sequence, the method 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, the making of the probes including making a minority of probes which have one or more sequence errors in them, errors being sequences not in the defined sequence;

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

(c) cooling the single stranded probes so that a pool of DNA duplexes are formed, the pool of duplexes formed including probes hybridized to their complete complementary probe and having normal topographical shape and other duplexes formed with at least one of the DNA probes in time duplex not of the defined sequence thus creating a duplex having an irregularity in its topographical shape;

(d) exposing the pool of DNA duplexes to a DNA binding agent which will selectively bind to DNA duplexes which have irregularities in their topographical shapes;

(e) separating out the DNA duplexes to which the DNA binding agent bound to leave DNA duplexes with the correct defined sequence;

(f) denaturing the DNA duplexes from the prior step to release the single stranded DNA probes from the DNA duplexes;

(g) cooling the DNA duplexes from the prior step 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 longer 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.

5. A method as claimed in claim 4 wherein the separation is performed by affinity binding the DNA binding agent at a fixed location.

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

7. A method, as claimed in claim 4 wherein steps (f) through (g) are performed repeatedly to assemble the entire pre-selected DNA sequence desired.