Detection of a nucleic acid sequence or a change therein

• US 5,856,092 A
• Filed: 04/27/1995
• Issued: 01/05/1999
• Est. Priority Date: 02/13/1989
• Status: Expired due to Term

• Current Assignee: Laboratory Corporation Of America Holdings
• Original Assignee: Geneco Pty., Ltd.
• Inventors: Timms, Peter, Walsh, Terence Patrick, Dale, James Langham
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/429,659
• Time in Patent Office: 1,349 Days
• Field of Search: 435/6, 435/91.1, 536/23.1, 536/24.33, 536/25.32, 536/25.4, 935/76, 935/77, 436/94
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism

Abstract

A method for detecting a specific polynucleotide sequence in a material is disclosed. The method includes exposing said material to an oligonucleotide primer having a sequence complementary to part of said specific polynucleotide sequence wherein said primer binds to part of said polynucleotide sequence when present in said material. Primer bound to the polynucleotide sequence is extended wherein any extended primer includes a detectable element and/or a separation element. Any extended primer is then separated into a fraction wherein said fraction does not have detectable element not included in said extended primer. One then determines whether any extended primer is present in said fraction by assaying said fraction for said extended primer wherein the presence of said extended primer is indicative of the presence of the specific polynucleotide sequence in said material and the absence of said extended primer is indicative of the presence of the specific polynucleotide sequences in said material and the absence of said extended primer in said fraction is indicative of the absence of the specific polynucleotide sequence in said material.

Claims

1. A method for detecting whether a specific nucleotide or base is at a particular position in a specific polynucleotide sequence comprising:

a) exposing, under hybridizing conditions, said specific polynucleotide sequence to an oligonucleotide primer wherein said primer has a sequence complementary to part of the specific polynucleotide sequence wherein said primer has incorporated at its 5'"'"' end an element selected from the group consisting of a separation element and a detectable element, and wherein said primer hybridizes at a location selected from the group consisting of (i) immediately adjacent to the particular position and (ii) not immediately adjacent to the particular position whereby there is an intervening sequence between the particular position and primer bound to the specific polynucleotide sequence;

b) extending said hybridized primer up to and including said specific nucleotide or base wherein the 3'"'"' terminal nucleotide is a ddNTP and further includes an element selected from the group consisting of a separation element and a detectable element with the proviso that said extended primer has at least one separation element and at least one detectable element;

c) separating the product of step b) into fractions wherein one said fraction contains the primer extension product that contains the chain terminating nucleotide at said particular position; and

d) determining whether said primer extension product having said chain terminating nucleotide at said particular position is present in said fraction by assaying said fraction wherein the assay does not include a digestion step.

2. The method of claim 1 wherein said separating step comprises:

contacting any extended primer with a molecule having affinity for the separation element, said molecule being linked to a support that facilitates said separating; and

separating said contacted extended primer to provide said fraction.

3. The method of claim 2 wherein said separation element and said molecule are selected from the group listed. as follows:

space="preserve" listing-type="tabular">______________________________________ Molecule Having AffinitySeparation Element for Separation Element______________________________________(a) a ligand for a specific receptor for which there is the ligand;

a specific receptor;

(b) a specific the ligand for the receptor;

specific receptor;

(c) an antigen;

a specific antibody of the antigen;

(d) a specific the antigen;

antibody for antigen;

(e) an a specific antibody of antiidiotypic the antiidiotypic antibody;

antibody;

(f) an antibody for an antiidiotypic antibody an specific for the antiidiotypic antibody;

antibody;

(g) a haptenic a specific antibody of group;

the haptenic group;

(h) a specific the haptenic group;

antibody for a haptenic group;

(i) an enzyme;

a tight binding inhibitor for the enzyme; and

(j) a tight binding the enzyme. inhibitor for an enzyme;

______________________________________

4. The method of claim 2 wherein said support is a test strip.

7. The method of any one of claims 1, 5, or 6 wherein said primer binds to part of said specific polynucleotide in said material immediately adjacent to the particular position.

8. The method of any one of claims 1, 5, or 6 wherein said primer binds to part of said specific nucleotide sequence in said material not immediately adjacent to the particular position whereby there is an intervening sequence between the particular position and primer bound to the specific polynucleotide sequence.

9. The method of any one of claims 1, 5, or 6 wherein the separation element comprises an inert support.

10. The method of any one of claims 1, 5, or 6 wherein the separation element comprises an inert solid support.

11. The method of any one of claims 1, 5, or 6 wherein said extending step comprises using four different nucleotides selected from the group consisting of dATP, dCTP, dGTP, dTTP, dUTP, dITP, ddATP, ddCTP, ddGTP, ddITP, and ddTTP, and wherein said nucleotides incorporate at least one element selected from the group consisting of a detectable element and a separation element.

12. The method of claim 11 wherein at least one of said nucleotides includes a detectable element.

13. The method of claim 11 wherein at least one of said nucleotides includes a separation element.

14. The method of claim 11 wherein at least one of said nucleotides includes separation and detectable elements.

15. The method of any one of claims 1, 5, or 6 wherein said extending step comprises using one nucleotide sequence selected from the group consisting of ddITP, ddATP, ddCTP, ddGTP, and ddTTP, and wherein said nucleotide incorporates at least one element selected from the group consisting of a detectable element and a separation element.

16. The method of claim 15 wherein said nucleotide includes a detectable element.

17. The method of claim 15 wherein said nucleotide includes a separation element.

18. The method of claim 15 wherein said nucleotide includes separation and detectable elements.

19. The method of any one of claims 1, 5, or 6 wherein said separating step comprises, contacting any extended primer with a molecule having affinity for said separation element;

and separating said contacted extended primer(s) to provide said fraction wherein said separation element and said molecule are selected from the group listed as follows;

space="preserve" listing-type="tabular">______________________________________ Molecule Having AffinitySeparation Element for Separation Element______________________________________(a) a ligand for which a receptor for the ligand;

there is a receptor;

a receptor;

the ligand for the receptor;

(c) an antigen;

an antibody for the antigen;

(d) an antibody for an the antigen;

antigen;

(e) an antiidiotypic an antibody for the antiidiotypic antibody;

antibody;

(f) an antibody for an an antiidiotypic antibody for the antiidiotypic antibody;

antibody;

(i) a haptenic group;

an antibody for the haptenic group;

(h) an antibody for a the haptenic group;

haptenic group;

(i) an enzyme;

a binding inhibitor for the enzyme;

and(j) a binding inhibitor the enzyme. for an enzyme;

______________________________________

20. The method of any one of claims 1, 5, or 6 wherein the specific polynucleotide sequence(s) is a DNA sequence.

21. The method of any one of claims 1, 5, or 6 wherein the specific polynucleotide sequence(s) is an RNA sequence.

22. The method of any one of claims 1, 5, or 6 wherein the specific polynucleotide sequence(s) is from an organism selected from the of infectious organisms and disease causing organisms.

23. The method of any one of claims 1, 5, or 6 wherein the oligonucleotide primer incorporates a detectable element at its 5'"'"' end.

24. The method of any one of claims 1, 5, or 6 wherein the separating step is a washing step.

5. A method for detecting whether the same or different specific nucleotides or bases are at particular positions in at least two different specific polynucleotide sequences comprising:

a) exposing, under hybridizing conditions, said specific polynucleotide sequences to at least two different oligonucleotide primers, wherein each of said different oligonucleotide primers has a sequence complementary to part of one of said different specific polynucleotide, sequences wherein;

each of said primers hybridizes to its complementary polynucleotide sequence when present in said material, each of said oligonucleotide primers hybridizing to part of a different specific polynucleotide sequence to that of the other primer(s),

each of said primers hybridizes to its complementary specific polynucleotide sequence in said material a location selected from the group consisting of (i) immediately adjacent to the particular position and (ii) not immediately adjacent to the particular position whereby there is an intervening sequence between the particular position and primer hybridized to the specific polynucleotide sequence, and

each of said primers has incorporated at their 5'"'"' end an element selected from the group consisting of a separation element and a detectable element;

b) extending said hybridized different oligonucleotide primers up to and including said specific nucleotide or base wherein the 3'"'"' terminal nucleotide is a ddNTP and further includes an element selected from the group consisting of a detectable element and a separation element, with the proviso that each of said extended primers has at least one separation element and at least on detectable element;

c) separating the product(s) of step b) into fractions wherein one said fraction contains the primer extension product(s) that contain the chain terminating nucleotide at said particular position; and

d) determining whether any of said primer extension product having said chain terminating nucleotide at said particular position is present in said fraction by assaying said fraction wherein the assay does not include a digestion step.

25. The method of claim 5 or claim 6 wherein each of said primers incorporates a different separation element.

26. The method of claim 5 or claim 6 wherein said separating step comprises:

contacting any extended primers with molecules having affinity for the separation elements, said molecules being linked to supports that facilitate said separating; and

separating said contacted extended primers to provide said fraction.

27. The method of claim 26 wherein said separation elements and said molecules are selected from the group listed as follows:

space="preserve" listing-type="tabular">______________________________________ Molecule Having AffinitySeparation Element for Separation Element______________________________________(a) a ligand for a specific receptor for which there is the ligand;

a specific receptor;

(b) a specific the ligand for the receptor;

specific receptor;

(c) an antigen;

a specific antibody of the antigen;

(d) a specific the antigen;

antibody for antigen;

(e) an a specific antibody of antiidiotypic the antiidiotypic antibody;

antibody;

(f) an antibody for an antiidiotypic antibody an specific for the antiidiotypic antibody;

antibody;

(g) a haptenic a specific antibody of group;

the haptenic group;

(h) a specific the haptenic group;

antibody for a haptenic group;

(i) an enzyme;

a tight binding inhibitor for the enzyme; and

(j) a tight binding the enzyme. inhibitor for an enzyme;

______________________________________

28. The method of claim 26 wherein said supports are test strips.

29. The method of claim 28 wherein said separating step comprises precipitating any extended primer(s) and resuspending said extended primer(s) in a solution conducive to binding of said separation element(s) to a molecule(s) having affinity for said separation element(s).

6. A screening method for detecting whether the same or different specific nucleotides or bases are at particular positions in at least two different specific polynucleotide sequences comprising:

a) exposing, under hybridizing conditions, said specific nucleotide sequences to at least two different oligonucleotide primers, each of said different oligonucleotide primers having a sequence complementary to part of one of said different specific polynucleotide sequences wherein;

each of said primers hybridize to its complementary polynucleotide sequence when present in said material, each of said oligonucleotide primers hybridizing to part of a different specific polynucleotide sequence to that of the other primer(s),

each of said primers hybridizes to its complementary specific polynucleotide sequence in said material at a location selected from the group consisting of (i) immediately adjacent to the particular position and (ii) not immediately adjacent to the particular position whereby there is an intervening sequence between the particular position and primer hybridized to the specific polynucleotide sequence when presenting said material; and

each of said primers has incorporated at their 5'"'"' end an element selected from the group consisting of a separation element and a detectable element;

b) extending said hybridized different oligonucleotide primers up to and including said specific nucleotide or bas e wherein the 3'"'"' terminal nucleotide is a ddNTP and further includes an element selected from the group consisting of a detectable element and a separation element with the proviso that each of said extended primers as at least one separation element and at least one detectable element;

c) separating the product(s) of step b) into fractions wherein one said fraction contains the primer extension product(s) that contain the chain terminating nucleotide at said particular position; and

d) determining whether at least one of said primers extension products having said chain terminating nucleotide at said particular position is present in said fraction by assaying said fraction wherein the assay does not include a digestion step.