Methods and compositions for the detection of sequences in selected DNA molecules

• US 5,137,806 A
• Filed: 12/11/1989
• Issued: 08/11/1992
• Est. Priority Date: 12/11/1989
• Status: Expired due to Term

• Current Assignee: Astrazeneca UK Limited (Astrazeneca PLC)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: LeMaistre, Anne, Lee, Ming-Shen
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): ESCALLON, MIGUEL H
• Application Number:
  US07/448,118
• Time in Patent Office: 974 Days
• Field of Search: 435/6, 435/91, 435/805, 435/948, 435/810, 435/803, 435/7, 436/27, 436/501, 436/811, 935/6, 935/17, 935/19, 935/78, 935/88
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

The present disclosure relates to novel procedures and primers for use in conenction with PCR or in vitro DNA sequence amplification to detect sequence variants, such as sequence modifications or mutations. The invention will have particular applicability in the detection of point or other relatively short mutations where the expected location or configuration of the mutation is known. Primers of the invention incorporate a 3'"'"' terminal nucleotide or nucleotides complementary to the sequence variance, and thereby serve to successfuly prime chain elongation only on DNA templates which include the particular variant. Exemplary mutations suitable for detection through practice of the invention include those involved in beta-thalassemia, sickle cell anemia, hemoglobin C disease, diabetes, acute intermittent porphyria, lung, breast, and colon cancers and others.

Claims

1. A method for detecting the presence or absence of a target DNA sequence within an identified region of a selected DNA molecule, wherein when the target is present in the region, it has an expected location and configuration therein, the method comprising the steps of:

(a) obtaining a nucleic acid primer molecule having a template binding region that is capable of hybridizing to a first strand of the selected DNA molecule at a binding position 3'"'"' of and adjacent to the expected location of such a target sequence within the DNA molecule, said template binding region of the primer molecule being capable of binding to said DNA molecule when the target sequence is present or absent, the primer molecule further including at its 3'"'"' terminus and adjacent the template binding region a target sequence complementary region comprised of at least one nucleotide complementary to a corresponding nucleotide of the target sequence, said primer being capable of priming polymerase chain extension when said target sequence is present on said DNA molecule, yet incapable of priming polymerase chain extension when the target sequence is absent; and

(b) determining the ability of the primer molecule to prime the polymerase chain extension using the selected DNA molecule as a template, to detect the presence or absence of the target sequence within the selected DNA molecule.

2. The method of claim 1, wherein determining the ability of the primer molecule to prime polymerase chain extension includes the steps of:

(a) obtaining a second nucleic acid primer molecule having a second template binding region that is capable of hybridizing to the second strand of the selected DNA molecule at a binding position 3'"'"' of the expected location of such a target sequence; and

(b) determining the ability of the first and second primers to prime polymerase chain reaction synthesis of both strands of the DNA molecule, such an ability being indicative of the presence of the target sequence within the selected DNA molecule.

3. The method of claim 2, wherein determining the ability of the first and second primers to prime polymerase chain reaction synthesis includes the steps of:

(a) hybridizing the primers with the selected DNA molecule to form primed templates;

(b) subjecting the primed templates to polymerase chain extension to form polymerase chain extended products; and

(c) detecting the generation of polymerase chain extended products having a size corresponding to about the cumulative size of the first and second primers and the distance between their respective primer binding positions along the DNA molecule.

4. The method of claim 3 wherein detecting the generation of polymerase chain extended products comprises subjecting the polymerase chain extended products to gel electrophoresis, and identifying products of the appropriate size.

5. The method of claim 4 wherein generated polymerase chain extended products are identified by means of a label.

6. The method of claim 2, further including determining the ability of the primer molecules to prime polymerase chain extension using a reference DNA molecule 42 as template, the reference molecule having such an identified region 44 which has been characterized in terms of the presence or absence of the target sequence.

7. The method of claim 1, wherein the primer molecule further includes, at a position 1 to 3 nucleotides 5'"'"' of the target sequence complementary region and positioned between the template binding region and the target sequence complementary region, a nonsense nucleotide that is not complementary to its corresponding nucleotide on the selected DNA molecule.

8. The method of claim 6, wherein the identified region of the selected and reference DNA molecule comprises a selected gene and the target DNA sequence comprises a mutation in said gene.

9. The method of claim 8, wherein the mutation comprises a sequence present in the selected DNA molecule and absent from the reference DNA molecule.

10. The method of claim 8, wherein the mutation comprises a sequence absent from the selected DNA molecule and present in the reference DNA molecule.

11. The method of claim 9 or 10, wherein the target sequence comprises a point mutation, and the target sequence complementary region of said primer comprises a nucleotide that is complementary to the point mutation.

12. The method of claim 9 or 10, wherein the target sequence comprises an insertion mutation, and the target sequence complementary region comprises at least one nucleotide that is complementary to the 3'"'"' nucleotide of the insertion.

13. The method of claim 9 further comprising the steps of:

(a) obtaining a third nucleic acid primer molecule having a third template binding region that is capable of hybridizing to a first or second strand of the selected DNA molecule at a binding position 3'"'"' of and adjacent to the expected location of such a target sequence within the DNA molecule, said template binding region of the primer molecule being capable of binding to said DNA molecule when the target sequence is either present or absent, the primer molecule further including at its 3'"'"' terminus and adjacent the template binding region at least one nucleotide complementary to the corresponding nucleotide on the reference DNA molecule, said primer being capable of priming polymerase chain extension when said target is present on the DNA molecule, yet incapable priming polymerase chain extension when the target sequence is absent; and

(b) determining the ability of the third primer molecule to prime the polymerase chain extension using the selected DNA molecule as a template, to detect the presence or absence of the target sequence within the selected DNA molecule.

14. The method of claim 13, wherein the third template binding region corresponds to the first template binding region.

15. The method of claim 13, wherein determining the ability of the third primer molecule to prime polymerase chain extension includes the steps of:

(a) obtaining a fourth nucleic acid primer molecule having a fourth template binding region that is capable of hybridizing to the opposite strand from the third primer molecule at a binding position 3'"'"' of the expected location of such a target sequence; and

(b) determining the ability of the third and fourth primers to prime polymerase chain extension on both strands of the reference or selected DNA molecule, such an ability being indicative of the absence of the target sequence within the respective DNA molecule.

16. The method of claim 15, wherein the fourth nucleic acid primer molecule corresponds to the second nucleic acid primer molecule.

17. A method for detecting the presence or absence of a point mutation within a selected gene of a selected DNA molecule, wherein when said point mutation is present in the gene, it has an expected location and configuration therein, the method comprising the steps of:

(a) obtaining a nucleic acid primer molecule having a template binding region that is capable of hybridizing to a first strand of the selected DNA molecule at a binding position 3'"'"' of and adjacent to the expected location of the point mutation, said template binding region of the primer molecule being capable of binding to said DNA molecule when the point mutation is either present or absent, the primer molecule further including at its 3'"'"' terminus and adjacent the template binding region a nucleotide that is complementary to the point mutation, said primer being capable of priming polymerase chain extension when said point mutation is present on said DNA molecule, yet incapable of priming polymerase chain extension when the point mutation is absent; and

(b) determining the ability of the primer molecule to prime polymerase chain extension using the selected DNA molecule as a template, to detect the presence or absence of the point mutation within the selected DNA molecule.

18. The method of claim 17, wherein determining the ability of the primer molecule to prime polymerase chain extension includes the steps of:

(a) obtaining a second nucleic acid primer molecule having a second template binding region that is capable of hybridizing to the second strand of the selected DNA molecule at a binding position 3'"'"' of the expected location of such a point mutation; and

(b) determining the ability of the first and second primes to prime polymerase chain reaction synthesis of both strands of the DNA molecule, such an ability being indicative of the presence of the point mutation in the expected configuration within the selected DNA molecule.

19. The method of claim 18, further including determining the ability of the first and second primer molecules to prime polymerase chain extension using a reference DNA molecule as template, the reference molecule known to include the gene without the point mutation.

20. A nucleic acid primer molecule for use in connection with polymerase chain extension for detecting the presence or absence of a target DNA sequence that has an expected location and configuration within an identified region of a selected DNA molecule, the nucleic acid primer molecule having a template binding region that is capable of hybridizing to a first strand of the selected DNA molecule at a binding position 3'"'"' of and adjacent to the expected location of such a target sequence within the DNA molecule, the primer molecule further including at its 3'"'"' terminus and adjacent the template binding region at target sequence complementary region comprised of at least one nucleotide complementary to a corresponding nucleotide of the target sequence.

21. The primer molecule of claim 20, further including, at a position 1 to 3 nucleotides 5'"'"' of the target sequence complementary region and positioned between the template binding region and the target sequence complementary region, a nonsense nucleotide that is not complementary to its corresponding nucleotide on the selected DNA molecule.

22. The primer molecule of claim 20, wherein the target sequence comprises a point mutation, and the target sequence complementary region of said primer comprises a nucleotide that is complementary to the point mutation.