Methods and devices for DNA sequencing and molecular diagnostics

Patent metadata

Field	Value
Publication number	US-9670540-B2
Application number	US-201214234011-A
Country	US
Kind code	B2
Filing date	Jul 23, 2012
Priority date	Jul 21, 2011
Publication date	Jun 6, 2017
Grant date	Jun 6, 2017

Abstract

The present invention is directed to methods for capturing, amplifying and identifying one or more of a plurality of target nucleotide sequences in a sample. The present invention is further directed to a device comprising a solid support having a plurality of wells or pillars and a plurality of oligonucleotides attached to the wells or pillars. Other aspects of the invention are directed to methods of making such devices.

First claim

What is claimed is:

1. A method for identifying one or more of a plurality of target nucleotide sequences in a sample comprising:
providing a sample potentially containing one or more target nucleotide sequences and/or complements thereof, each target nucleotide sequence comprising a first, second, and third target portion, wherein the second target portion is between the first and third target portions;
providing one or more oligonucleotide primer sets, each set characterized by (a) a first oligonucleotide primer comprising a first primer portion complementary to the first target portion and a second primer portion that is the same as the second target portion, wherein the second primer portion is 5′ to the first primer portion, and (b) a second oligonucleotide primer comprising a third primer portion that is the same as the third target portion;
providing a first polymerase;
blending the sample, the oligonucleotide primer sets, and the first polymerase to form a polymerase extension reaction mixture;
subjecting the polymerase extension reaction mixture to a hybridization treatment, wherein the first and third primer portions of the first and second oligonucleotide primers, respectively, hybridize to their respective complementary portions of the target nucleotide sequence or complement thereof, and an extension treatment, wherein the hybridized oligonucleotide primers extend to form primary primer extension products;
denaturing the primary primer extension products from the target nucleotide sequences and complements thereof;
providing a second polymerase;
blending the polymerase extension reaction mixture after said subjecting it to the hybridization treatment and the extension treatment and the second polymerase to form a polymerase amplification reaction mixture;
subjecting the polymerase amplification reaction mixture to one or more polymerase amplification reaction cycles comprising a denaturation treatment, wherein hybridized nucleic acid sequences are separated, a hybridization treatment, wherein the first and third primer portions of the first and second oligonucleotide primers, respectively, hybridize to their respective complementary portions of the primary primer extension products and an extension treatment, wherein hybridized oligonucleotide primers extend to form first oligonucleotide primer extension products and second oligonucleotide primer extension products; and
subjecting the first and/or second oligonucleotide primer extension products to a sequencing reaction to identify the presence of one or more target nucleotide sequences in the sample.

2. The method of claim 1 , wherein said providing a sample comprises:
appending one or more first, second, and/or third target portions, or complements thereof, to remaining parts of the one or more target nucleotide sequences, or complements thereof.

3. The method of claim 2 , wherein the first, second, and third target portions, or complements thereof, are appended to the remaining parts of the one or more target nucleotide sequences, or complements thereof, using an enzyme selected from the group consisting of ligases, polymerases, recombinases, terminal transferases, endonucleases, DNA repair enzymes, and reverse transcriptases.

4. The method of claim 1 , wherein one or both of the first and second oligonucleotide primers in each primer set is attached to a solid support.

5. The method of claim 4 further comprising:
repeating one or more times said providing a second polymerase, said blending to form a polymerase amplification reaction mixture, and said subjecting the polymerase amplification reaction mixture to one or more polymerase amplification reaction cycles, to amplify the first and second oligonucleotide primer extension products prior to said subjecting the first and/or second oligonucleotide primer extension products to the sequencing reaction to identify the presence of one or more target nucleotide sequences in the sample.

6. The method of claim 4 , wherein the solid support comprises a plurality of pillars.

7. The method of claim 6 , wherein one or both of the oligonucleotide primers in an oligonucleotide primer set are attached to the pillar surfaces, but not to surfaces of the solid support between the pillars.

8. The method of claim 1 further comprising:
denaturing the target nucleotide sequences from their complements in the sample prior to said subjecting the polymerase extension reaction mixture to the hybridization and the extension treatment.

9. The method of claim 5 , wherein the second polymerase exhibits strand displacement activity.

10. The method of claim 5 , wherein the first oligonucleotide primer extension products comprise the first primer portion, the second primer portion, a third portion that is complementary to the third primer portion, and a fourth portion that is complementary to the second primer portion.

11. The method of claim 5 , wherein the second oligonucleotide primer extension products comprise a first portion that is complementary to the first primer portion, a second portion that is complementary to the second primer portion, the third primer portion, and a fourth portion that is the same as the second primer portion, wherein the fourth portion is 5′ to the second portion.

12. The method of claim 11 , wherein the second oligonucleotide primer extension products hairpin by hybridization between the second and fourth portions, said hairpin extending from its 3′ end to form full-length hair-pinned second oligonucleotide primer extension products.

13. The method of claim 12 , wherein the first primer portion of the first oligonucleotide primers hybridize to their complementary sequence on the full-length hair-pinned second oligonucleotide primer extension products and extend to make first oligonucleotide primer extension products while displacing part of the full-length hair-pinned second oligonucleotide primer extension products having the same sequence.

14. The method of claim 13 further comprising:
cleaving second oligonucleotide primer extension products from the solid support.

15. The method of claim 14 further comprising:
providing third oligonucleotide primers, each third oligonucleotide primer comprising a fifth primer portion complementary to a fifth portion of the first oligonucleotide primer extension products, wherein said third oligonucleotide primer is attached to the solid support;
providing fourth oligonucleotide primers, each fourth oligonucleotide primer comprising a sequence complementary to a region of the first oligonucleotide primer extension product;
providing a polymerase and a ligase;
blending the first oligonucleotide primer extension products, the third oligonucleotide primers, the fourth oligonucleotide primers, the polymerase, and the ligase to form an extension-ligation mixture;
subjecting the extension-ligation mixture to a hybridization treatment, wherein the third and fourth oligonucleotide primers hybridize to their complementary regions on the first oligonucleotide primer extension products, and an extension-ligation treatment, wherein the hybridized fourth oligonucleotide primers extend to form complements of a portion of the first oligonucleotide primer extension product and ligate to the fifth primer portion of hybridized third oligonucleotide primers to form ligation extension products; and
cleaving the first oligonucleotide primer extension products from the solid support, wherein said ligation extension products are subjected to said sequencing reaction to identify the presence of one or more target nucleotide sequences in the sample.

Assignees

• Barany Francis A
• Soper Steven A
• Grills George
• Cheng Yu-Wei
• Huang Jianmin
• Wang Hong
• Witek Malgorzata A
• Park Daniel Sang-Won
• Murphy Michael C
• Mccarley Robin Lindsey
• Hupert Mateusz L
• Univ Cornell
• Univ Louisiana State

Inventors

• Barany Francis A
• Soper Steven A
• Grills George
• Cheng Yu-Wei
• Huang Jianmin
• Wang Hong
• Witek Malgorzata A
• Park Daniel Sang-Won
• Murphy Michael C
• Mccarley Robin Lindsey
• Hupert Mateusz L

Classifications

• C12Q2563/131

  the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin · CPC title

• C12Q2565/543

  characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2525/197

  incorporating a spacer/coupling moiety · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title