Methods for solid-phase amplification of DNA template (SPADT) using multiarrays

• US 6,221,635 B1
• Filed: 05/06/1999
• Issued: 04/24/2001
• Est. Priority Date: 05/06/1999
• Status: Active Grant

• Current Assignee: The Wistar Institute
• Original Assignee: The Wistar Institute
• Inventors: Mukhopadhyay, Sunil, Rovera, Giovanni
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): SWITZER, JULIET CAROLINE
• Application Number:
  US09/306,290
• Time in Patent Office: 719 Days
• Field of Search: 435/91.2, 435/6, 435/5
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The present invention relates to a novel method of detecting specific nucleic acids in a biological sample using solid-phase amplification of DNA template (SPADT) using multiarrays.

Claims

1. A method of detecting the presence or absence of a specific nucleic acid in a sample comprising DNA, said method comprising performing an amplification reaction, wherein a solid support on which a 5′

and a 3′

primer are irreversibly bound and said DNA is absorbed such that it is reversibly bound, is incubated under amplification conditions, and determining whether said specific nucleic acid is amplified, wherein when said specific nucleic acid is amplified, said specific nucleic acid is present in said sample and when said specific nucleic acid is not amplified, said specific nucleic acid is not present in said sample.

2. The method of claim 1, wherein said solid support comprises a solid to which said DNA cannot be irreversibly bound upon adding said DNA to said solid support in the absence of any further reaction.

3. The method of claim 2, wherein said solid support is selected from the group consisting of a nylon membrane, and a nitrocellulose membrane.

4. The method of claim 3, wherein said solid support is a nylon membrane.

5. The method of claim 1, wherein said primers are irreversibly bound to said solid support by UV-crosslinking said primers to said support.

6. The method of claim 1, wherein said 5′

and 3′

primers further comprise a poly-thymidine sequence at their 5′

ends.

7. The method of claim 6, wherein said poly-thymidine sequence comprises from about 15 to about 80 thymidine nucleotides.

8. The method of claim 7, wherein said 5′

and 3′

primers comprise a sequence homologous to the sequence of said specific nucleic acid, said primers ranging in length from about 19 nucleotides to about 23 nucleotides.

9. The method of claim 1, wherein when said primers are in double stranded form, the thermal melting temperature of said double stranded form of said primers is in the range of about 55°

C. to about 65°

C.

10. The method of claim 8, wherein said 5′

and 3′

primers are lacking a GC rich region at the 3′

end of said 5′ and

3′

primers.

11. The method of claim 1, wherein said amplification is performed by a reaction selected from the group consisting of a polymerase chain reaction, and a ligase reaction.

12. The method of claim 1, wherein said determination of whether said specific nucleic acid is amplified is performed by incorporating a detectable label into said amplified nucleic acid during amplification and detecting said label.

13. The method of claim 1, wherein said amplification conditions comprise about 2 mM MgCl₂ and about 50 mM KCl.

14. The method of claim 1, wherein said specific nucleic acid is selected from the group consisting of a nucleic acid comprising a mutation, a nucleic acid comprising a mutation in a cancer gene, and a nucleic acid comprising a sequence present in the genome of an infectious agent.

15. A method of determining the presence or absence of expression of a specific nucleic acid in a cell, said method comprising reverse transcribing RNA obtained from said cell to produce DNA, performing an amplification reaction, wherein a solid support on which a 5′

and a 3′

primer are irreversibly bound and said DNA is adsorbed such that it is reversibly bound is incubated under amplification conditions, and determining whether said DNA is amplified, wherein when said DNA is amplified, said specific nucleic acid is expressed in said cell and when said DNA is not amplified, said specific nucleic acid is not expressed in said cell.

16. A method of determining the presence or absence of a specific RNA molecule in a sample, said method comprising reverse transcribing said RNA to DNA, performing an amplification reaction wherein a solid support on which a 5′

and a 3′

primer are irreversibly bound and said DNA is adsorbed such that it is reversibly bound is incubated under amplification conditions, and determining whether said DNA is amplified, wherein when said DNA is amplified, said specific RNA is present in said sample, and when said DNA is not amplified, said specific RNA is not present in said sample.

17. The method of claim 16, wherein said specific RNA is viral RNA.

18. A method of designing a primer which amplifies a specific nucleic acid provided said specific nucleic acid is present in a sample comprising DNA, said method comprising

(a) synthesizing at least one 5′

and one 3′

primer homologous to said specific nucleic acid;

(b) performing an amplification reaction wherein a solid support on which said primer is irreversibly bound and said DNA is absorbed such that it is reversibly bound is incubated under amplification conditions to generate an amplified nucleic acid;

(c) determining the specificity of said primer by comparing the ability of said primer to produce a detectable amount of said amplified nucleic acid in said amplification reaction in the presence or absence of said specific nucleic acid, wherein a detectable amount of amplified nucleic acid in said amplification reaction in the presence of said specific nucleic acid, compared with the absence of a detectable amount of said amplified nucleic acid in the absence of said specific nucleic acid, is an indicator that said primer is specific for ampliyfing said specific nucleic acid;

(d) comparing the characteristics of said primers of step (c) which are specific for said specific nucleic acid to identify common features shared among the most specific of said primers; and

(e) synthesizing an additional primer incorporating said features identified in step (d), thereby designing a primer which amplifies a nucleic acid provided said specific nucleic acid is present in a sample comprising DNA.

19. The method of claim 18, wherein said comparing step (d) is performed using an algorithm.