Method of Detecting Genetic Mutations

• US 20090111108A1
• Filed: 04/10/2007
• Published: 04/30/2009
• Est. Priority Date: 04/10/2006
• Status: Abandoned Application

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Zhu, Jun, Gao, Feng
• Application Number:
  US12/226,139
• Publication Number:
  US 20090111108A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism

Abstract

The present invention relates, in general, to drug resistance, and, in particular, to a method of detecting drug resistance populations, including minor drug resistance viral populations.

Claims

1. A method of detecting, or determining the frequency of, a mutation or nucleotide modification in a multiplicity of nucleic acid templates comprising:

i) embedding a multiplicity of nucleic acid templates and modified 3′

amplification primers or modified 5′

amplification primers in a semi-solid support under conditions such that said modified amplification primers are immobilized in said semi-solid support;

ii) contacting said semi-solid support resulting from step (i) with a polymerase, deoxynucleotide triphosphates (dNTPs), and unmodified 5′

or 3′

amplification primers;

iii) treating the composition resulting from step (ii) under conditions such that said modified and/or unmodified amplification primers hybridize to said nucleic acid templates to form primed templates;

iv) effecting amplification of said primed templates in said semi-solid support, wherein double-stranded products of said amplification localize around said primed templates;

v) denaturing said double-stranded amplification products and removing resulting single strands not immobilized in said semi-solid support;

vi) hybridizing sequencing primers to said immobilized single stands resulting from step (v) and effecting extension of said hybridized sequencing primers in the presence of at least two nucleotides bearing different detectable labels to form extension products, wherein one of said nucleotides is incorporated into said extension product when said immobilized single strand bears said mutation or nucleotide modification and the other of said nucleotides is incorporated into said extension product when said immobilized single strand does not bear said mutation or modified nucleotide; and

vii) detecting the extension products of the sequencing primers resulting from step (vi), and determining the presence of, or the frequency of, said nucleic acid templates bearing said mutated or modified nucleotide.

2. The method according to claim 1 wherein said method is a method of determining the frequency of a mutation.

3. The method according to claim 1 wherein said multiplicity of nucleic acid templates comprise double-stranded DNA.

4. The method according to claim 1 wherein said multiplicity of nucleic acid templates are derived from a pathogen.

5. The method according to claim 4 wherein said pathogen is a viral or bacterial pathogen.

6. The method according to claim 5 wherein said pathogen is human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) or Mycobacterium tuberculosis (TB).

7. The method according to claim 5 wherein said pathogen is a drug resistant pathogen.

8. The method according to claim 1 wherein said multiplicity of nucleic acid templates are derived from genomes from cancer cells.

9. The method according to claim 8 wherein said cancer cells are human cancer cells.

10. The method according to claim 1 wherein said cancer cells are derived from a biopsy tissue, from blood or from a discharged material.

11. The method according to claim 10 wherein said discharged material comprises urine, feces or mucous or serous secretion.

12. The method according to claim 1 wherein said modified amplification primers are covalently attached to said semi-solid support.

13. The method according to claim 12 wherein said semi-solid support is an acrylamide gel.

14. The method according to claim 1 wherein unmodified 5′

primers and modified 3′

primers are embedded in said semi-solid support.

15. The method according to claim 1 wherein unmodified 3′

primers and modified 5′

primers are embedded in said semi-solid support.

16. The method according to claim 1 wherein said modified primers are acrydited primers.

17. The method according to claim 1 wherein said detectable labels are fluorescent labels.

18. A method of determining viral load in a patient comprising:

i) extracting viral DNA from a biological sample from said patient;

ii) embedding said DNA and modified 3′

amplification primers or modified 5′

amplification primers in a semi-solid support under conditions such that said modified amplification primers are immobilized in said semi-solid support;

iii) contacting said semi-solid support resulting from step (i) with a polymerase, deoxynucleotide triphosphates (dNTPs), and unmodified 5′

or 3′

amplification primers;

iv) treating the composition resulting from step (iii) under conditions such that said modified and/or unmodified amplification primers hybridize to said DNA to form primed templates,

v) effecting amplification of said primed templates in said semi-solid support, wherein double-stranded products of said amplification localize around said primed templates;

vi) denaturing said double-stranded amplification products and removing resulting single strands not immobilized in said semi-solid support;

vii) hybridizing sequencing primers to said immobilized single stands resulting from step (vi) and effecting extension of said hybridized sequencing primers in the presence of at least one nucleotide bearing a detectable label to form extension products; and

viii) detecting labeled extension products resulting from step (vii), and thereby determining said viral load.

19. The method according to claim 18 wherein, prior to step (ii), said DNA is sheared.

20. The method according to claim 19 wherein said sheared DNA is about 100 bases to about 1000 kb in length.

21. The method according to claim 20 wherein said sheared DNA is about 1 kb to about 100 kb in length.

22. A method of determining viral load in a patient comprising:

i) extracting viral RNA from a biological sample from said patient;

ii) reverse transcribing said RNA to produce cDNA and embedding said cDNA and modified 3′

amplification primers or modified 5′

amplification primers in a semi-solid support under conditions such that said modified amplification primers are immobilized in said semi-solid support;

iii) contacting said semi-solid support resulting from step (i) with a polymerase, deoxynucleotide triphosphates (dNTPs), and unmodified 5′

or 3′

amplification primers;

iv) treating the composition resulting from step (iii) under conditions such that said modified and/or unmodified amplification primers hybridize to said cDNA to form primed templates,

v) effecting amplification of said primed templates in said semi-solid support, wherein double-stranded products of said amplification localize around said primed templates;

vi) denaturing said double-stranded amplification products and removing resulting single strands not immobilized in said semi-solid support;

vii) hybridizing sequencing primers to said immobilized single stands resulting from step (vi) and effecting extension of said hybridized sequencing primers in the presence of at least one nucleotide bearing a detectable label to form labeled extension products; and

viii) detecting labeled extension products resulting from step (vii), and thereby determining said viral load.

23. A method of determining viral load in a patient comprising:

i) extracting viral DNA from a biological sample from said patient;

ii) embedding said DNA and modified 3′

amplification primers or modified 5′

amplification primers in a semi-solid support under conditions such that said modified amplification primers are immobilized in said semi-solid support;

iii) contacting said semi-solid support resulting from step (i) with a polymerase, deoxynucleotide triphosphates (dNTPs), and unmodified 5′

or 3′

amplification primers;

iv) treating the composition resulting from step (iii) under conditions such that said modified and/or unmodified amplification primers hybridize to said DNA to form primed templates,

v) effecting amplification of said primed templates in said semi-solid support, wherein double-stranded products of said amplification localize around said primed templates;

vi) denaturing said double-stranded amplification products and removing resulting single strands not immobilized in said semi-solid support;

vii) hybridizing a labeled probe specific for said virus to said immobilized single stands resulting from step (vi) to form a labeled duplex; and

viii) detecting the presence of said labeled duplex, and thereby determining said viral load.

24. The method according to claim 23 wherein, prior to step (ii), said DNA is sheared.

25. A method of determining viral load in a patient comprising:

i) extracting viral RNA from a biological sample from said patient;

ii) reverse transcribing said RNA to form cDNA and embedding said cDNA and modified 3′

amplification primers or modified 5′

amplification primers in a semi-solid support under conditions such that said modified amplification primers are immobilized in said semi-solid support;

iii) contacting said semi-solid support resulting from step (i) with a polymerase, deoxynucleotide triphosphates (dNTPs), and unmodified 5′

or 3′

amplification primers;

iv) treating the composition resulting from step (iii) under conditions such that said modified and/or unmodified amplification primers hybridize to said cDNA to form primed templates,

v) effecting amplification of said primed templates in said semi-solid support, wherein double-stranded products of said amplification localize around said primed templates;

vi) denaturing said double-stranded amplification products and removing resulting single strands not immobilized in said semi-solid support;

vii) hybridizing a labeled probe specific for said virus to said immobilized single stands resulting from step (vi) to form a labeled duplex; and

viii) detecting the presence of said labeled duplex, and thereby determining said viral load.

26. A method of detecting a mutation or nucleotide modification in a multiplicity of nucleic acid templates derived from DNA isolated from a biological sample comprising:

i) embedding said multiplicity of nucleic acid templates and modified 3′

amplification primers or modified 5′

amplification primers in a semi-solid support under conditions such that said modified amplification primers are immobilized in said semi-solid support;

ii) contacting said semi-solid support resulting from step (i) with a polymerase, deoxynucleotide triphosphates (dNTPs), and unmodified 5′

or 3′

amplification primers;

iii) treating the composition resulting from step (ii) under conditions such that said modified and/or unmodified amplification primers hybridize to said nucleic acid templates to form primed templates,

iv) effecting amplification of said primed templates in said semi-solid support, wherein double-stranded products of said amplification localize around said primed templates;

v) denaturing said double-stranded amplification products and removing resulting single strands not immobilized in said semi-solid support;

vi) hybridizing sequencing primers to said immobilized single stands resulting from step (v) and effecting extension of said hybridized sequencing primers in the presence of a nucleotide bearing a detectable label, wherein said nucleotide bearing said detectable label is incorporated into said extension product when said immobilized single strand bears said mutation or nucleotide modification so that a labeled extension product is formed; and

viii) detecting labeled extension products resulting from step (vii), and thereby determining the presence of said mutation or nucleotide modification.

27. The method according to claim 26 wherein said biological sample comprises DNA from cancer cells.