Method for the detection of genetic diseases and gene sequence variations by single nucleotide primer extension

• US 5,846,710 A
• Filed: 08/06/1993
• Issued: 12/08/1998
• Est. Priority Date: 11/02/1990
• Status: Expired due to Term

• Current Assignee: Laboratory Corporation Of America Holdings
• Original Assignee: St. Louis University
• Inventors: Bajaj, S. Paul
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/103,408
• Time in Patent Office: 1,950 Days
• Field of Search: 435/6, 435/91, 435/91.1, 435/91.2, 435/183, 536/23.1, 536/24.3, 536/24.33, 536/25.4, 935/6, 935/17, 935/19, 935/78, 935/80, 935/76, 935/77
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators

Abstract

Method for screening a sample oligonucleotide for a variation in sequence at a predetermined position thereof relative to a nucleic acid the sequence of which is known, wherein the sample oligonucleotide is provided as a single stranded molecule, the single stranded molecule is mixed with an inducing agent, a labeled nucleotide, and a primer having a sequence identical to a region flanking the predetermined position to form a mixture, the mixture having an essential absence of nucleotides constituted of bases other than the base of which the labeled nucleotide is constituted, the mixture is subjected to conditions conducive for the annealing of the primer to the single stranded molecule and the formation of a primer extension product incorporating the labeled nucleotide, and the mixture is analyzed for the presence of primer extension product containing labeled nucleotide.

Claims

1. A method for screening a DNA fragment of a gene for variation of nucleotide sequence at a predetermined position relative to the nucleotide sequence of the corresponding wild-type gene, the sequence of the wild-type gene being known at the predetermined position, the method comprising the steps of:

(a) providing the DNA fragment as a single stranded molecule,

(b) mixing the single stranded molecule with an inducing agent, an unlabeled primer having a nucleotide sequence complementary to a region flanking the predetermined position, and a labeled nucleotide to form a mixture, the mixture having an essential absence of nucleotides constituted of bases other than the base of which the labeled nucleotide is constituted,

(c) subjecting the mixture to conditions conducive for the annealing of the primer to the single stranded molecule and the formation of a primer extension product incorporating the labeled nucleotide,

(d) after step, analyzing the mixture for the presence or absence of primer extension product incorporating the labeled nucleotide, the analysis being carried out under conditions such that any primer which did not form a primer extension product incorporating the labeled nucleotide in step (c) is present throughout the analysis, and

(e) determining whether the sequence of the DNA fragment at the predetermined position is the same as or a variant of that of the wild-type gene based upon the presence or absence of the labeled nucleotide in the primer.

2. The method of claim 1 wherein the DNA fragment is being screened for a mutation relative to the wild-type gene at the predetermined position and wherein prior to step the sample DNA fragment is separated into at least two aliquots and then in step (b), labeled nucleotide corresponding to the sequence of the wild-type gene at the predetermined position is mixed with one of the aliquots to form a first mixture and labeled nucleotide corresponding to a mutation of the wild-type gene at the predetermined position is mixed with another one of the aliquots to form a second mixture.

3. The method of claim 1 wherein the sample DNA fragment is being screened for a mutation responsible for cystic fibrosis.

4. The method of claim 1 wherein the sample DNA is being screened for a mutation responsible for hemophilia B.

5. The method of claim 1 wherein prior to step (b) the sample DNA fragment is separated into at least two aliquots and then in step (b), labeled nucleotide corresponding to the sequence of a first variant of the wild-type gene at the predetermined position is mixed with one of the aliquots to form a first mixture and labeled nucleotide corresponding to a second variant of the wild-type gene at the predetermined position is mixed with another one of the aliquots to form a second mixture.

6. A method for screening an organism for genetic diseases or gene sequence variation resulting from a nucleotide substitution, translocation, insertion or deletion at a predetermined position, the sequence in the corresponding wild-type gene and normal variations thereof at that position being known, the method comprising

(a) providing a sample DNA fragment from the genome of that organism which contains the predetermined position,

(b) providing the sample DNA fragment as a single stranded molecule,

(c) mixing the single stranded molecule with an inducing agent, a primer having a nucleotide sequence complementary to a region flanking the predetermined position, and a labeled nucleotide to form a mixture, the mixture having an essential absence of nucleotides constituted of bases other than the base of which the labeled nucleotide is constituted,

(d) subjecting the mixture to conditions conducive for the formation of double stranded hybrids comprising the primer and the single stranded molecule and the formation of a primer extension product incorporating the labeled nucleotide,

(e) denaturing the double stranded hybrids formed in the mixture in step (d),

(f) analyzing the denatured double stranded hybrids for the presence of primer extension product incorporating labeled nucleotide, and

(g) determining whether the sequence of the DNA fragment at the predetermined position is the same as or a variant of the wild-type gene.

7. The method of claim 6 wherein the sample DNA fragment is being screened for a mutation relative to the wild-type gene at the predetermined position and wherein prior to step (c) the sample DNA fragment is separated into at least two aliquots and then in step (c), labeled nucleotide corresponding to the sequence of the wild-type gene at the predetermined position is mixed with one of said at least two aliquots to form a first mixture and labeled nucleotide corresponding to a mutation of the wild-type gene at the predetermined position is mixed with another one of said at least two aliquots to form a second mixture.

8. The method of claim 6 wherein the organism is being screened for cystic fibrosis.

9. The method of claim 6 wherein the organism is being screened for hemophilia B.

10. The method of claim 6 wherein prior to step (c) the sample DNA fragment is separated into at least two aliquots and then in step (c), labeled nucleotide corresponding to the sequence of a first variant of the wild-type gene at the predetermined position is mixed with one of said at least two aliquots to form a first mixture and labeled nucleotide corresponding to a second variant of the wild-type gene at the predetermined position is mixed with another one of said at least two aliquots to form a second mixture.

11. A method for comparing the nucleotide sequence of a DNA fragment of a gene at a predetermined position relative to the nucleotide sequence of the corresponding wild-type gene, the sequence of the wild-type gene being known, the method consisting essentially of the steps of:

(a) providing the DNA fragment as a single stranded molecule in solution,

(b) mixing the single stranded molecule with an inducing agent, a primer having a nucleotide sequence complementary to a region flanking the predetermined position, and a labeled nucleotide to form a mixture, the mixture having all essential absence of nucleotides constituted of bases other than the base of which the labeled nucleotide is constituted,

(c) subjecting the mixture to conditions conducive for the annealing of the primer to the single stranded molecule and the formation of a primer extension product incorporating the labeled nucleotide,

(d) after step (c), subjecting the mixture to gel electrophoresis under denaturing conditions, and

(e) determining whether the sequence of the DNA fragment at the predetermined position is the same as or a variant of the wild-type gene.

12. The method of claim 11 wherein the DNA fragment is being screened for a mutation relative to the wild-type gene at the predetermined position and wherein prior to step (b) the sample DNA fragment is separated into at least two aliquots and then in step (b), labeled nucleotide corresponding to the sequence of the wild-type gene at the predetermined position is mixed with one of the aliquots to form a first mixture and labeled nucleotide corresponding to a mutation of the wild-type gene at the predetermined position is mixed with another one of the aliquots to form a second mixture.

13. The method of claim 11 wherein the sample DNA fragment is being screened for a mutation responsible for cystic fibrosis.

14. The method of claim 11 wherein the sample DNA is being screened for a mutation responsible for hemophilia B.

15. The method of claim 11 wherein prior to step (b) the sample DNA fragment is separated into at least two aliquots to and then in step (b), labeled nucleotide corresponding to the sequence of a first variant of the wild-type gene at the predetermined position is mixed with one of the aliquots to form a first mixture and labeled nucleotide corresponding to a second variant of the wild-type gene at the predetermined position is mixed with another one of the aliquots to form a second mixture.

16. A method for screening a DNA fragment of a gene for variation of nucleotide sequence at a predetermined position relative to the nucleotide sequence of the corresponding wild-type gene, the sequence of the wild-type gene being known at the predetermined position, the method comprising:

(a) providing the DNA fragment as a single stranded molecule;

(b) mixing the single stranded molecule with an inducing agent, an unlabeled primer having a nucleotide sequence complementary to a region flanking the predetermined position, and a labeled nucleotide to form a mixture, the mixture having an essential absence of nucleotides that would eliminate the specificity of a primer extension reaction of step (c);

(c) subjecting the mixture to conditions conducive for annealing of the primer to the single stranded molecule and the formation of a primer extension product incorporating the labeled nucleotide at a position complementary to the predetermined position;

(d) after step (c), analyzing the mixture for the presence or absence of primer extension product incorporating the labeled nucleotide, the analysis being carried out under conditions such that any primer which did not form a primer extension product incorporating the labeled nucleotide in step (c) is present throughout the analysis; and

(e) determining whether the sequence of the DNA fragment at the predetermined position is the same as or a variant of that of the wild-type gene based upon the presence or absence of the labeled nucleotide in the primer.

17. The method of claim 16 wherein the mixture formed in step (b) has an essential absence of deoxynucleotides constituted of bases other than the base of which the labeled nucleotide is constituted.

18. The method of claim 16 wherein the labeled nucleotide is a deoxynucleotide.

19. The method of claim 16 wherein the labeled nucleotide is a deoxynucleotide and the mixture formed in step (b) has an essential absence of deoxynucleotides constituted of bases other than the base of which the labeled deoxynucleotide is constituted.

20. The method of claim 16 wherein the primer has a nucleotide sequence complementary to a region immediately flanking the predetermined position.