Method of gene mapping

• US 5,102,785 A
• Filed: 04/25/1988
• Issued: 04/07/1992
• Est. Priority Date: 09/28/1987
• Status: Expired due to Term

• Current Assignee: NEN Life Science Products Inc (Revvity, Inc.)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Livak, Kenneth J., Brenner, Sydney
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US07/185,741
• Time in Patent Office: 1,443 Days
• Field of Search: 435/6.91, 435/5, 436/94, 436/501, 436/63, 935/77, 935/78, 536/26, 536/27, 536/28, 536/29
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The method described characterizes each DNA segment to be mapped by cleaving it to produce DNA fragments which are then end labeled with a reporter(s) specific to the end nucleotides of each fragment. The labeled fragments are again cleaved to produce short fragments which are separated according to size. The short fragments are analyzed as to report identify and size which is indicative of the character of each fragment. By derivatizing the cleaved ends of the primary cleaved fragments, the labeling may be delayed until the second cleavage. Prior to the labeling the derivatized fragments, all underivatized fragments are removed, the derivatized fragments being immobilized.

Claims

1. A method for mapping a DNA segment made up of duplex strands of nucleotides by

cleaving the segment with a first restriction enzyme to produce fragments of DNA, each having a cleaved end,

attaching a reporter specific to a cleaved end nucleotide in each fragment,

cleaving the DNA fragments with a second restriction enzyme to produce short fragments,

separating the short fragments according to size, and

analyzing the short, separated fragments for the presence of reporters, the size and reporter identity being indicative of the character of each DNA fragment.

2. The method as set forth in claim 1 wherein the segment cleaving produces fragments of DNA with each cleaved end having one strand with a residue of nucleotides and a corresponding recessed strand and includes the step of attaching a reporter labeled nucleotide to the recessed strand to form a base pair with at least one residue nucleotide, the reporter being specific to each type of nucleotide.

3. The method as set forth in claim 2 wherein a third enzyme is used in conjunction with and in addition to the first enzyme or second enzyme to cleave different length fragments.

4. The method as set forth in claim 1 wherein a third enzyme is used in conjunction with and in addition to the first enzyme or second enzyme to cleave different length DNA fragments.

5. The method as set forth in claim 3 wherein the analysis is accomplished in real time.

6. The method of claim 2 wherein the segment cleaving step produces DNA fragments having a 5'"'"' overhang residue of nucleotides.

7. The method of claim 2 wherein the segment cleaving step produces DNA fragments having a 3'"'"' overhang residue of nucleotides.

8. The method of claim 7 wherein the reporter attachment is accomplished by using a polymerase exhibiting 3'"'"' exonuclease activity.

9. The method of claim 8 wherein the polymerase is selected from the group consisting of DNA polymerase I, T7 DNA polymerase, and T4 DNA polymerase.

10. The method of claim 2 wherein the segment cleaving step produces DNA fragments having blunt ends.

11. A method of mapping DNA segments made up of duplex strands of nucleotides using a reporter comprising:

reacting a mixture of DNA segments and a first restriction enzyme that cleaves the DNA to provide DNA fragments each with 5'"'"' or 3'"'"' overhang strands and a corresponding 3'"'"' or 5'"'"' recessed strand or with blunt ends,

incubating the DNA fragments with a DNA polymerase and a mixture of unlabeled deoxynucleotides and specific reporter labeled dideoxynucleotides to add nucleotides to the recessed strands complementary to the nucleotides in the overhang strands,

reacting the incubated DNA fragments with a second restriction enzyme to produce short DNA fragments some of which include the reporter labeled dideoxynucleotides and hence are reporter labeled,

separating the short DNA fragments according to size, and

detecting a reporter for each labeled fragment thereby identifying the 3'"'"' terminal added dideoxynucleotides.

12. The method as set forth in claim 11 which includes the step of:

correlating the size and reporter identification of each labeled short DNA fragment of the DNA segment.

13. The method as set forth in claim 12 wherein the ratio of labeled dideoxynucleotides to unlabeled deoxynucleotides is varied inversely according to the number of nucleotides in the overhang strands.

14. The method as set forth in claim 11 wherein a third enzyme is used in conjunction with and in addition to the first enzyme or second enzyme to cleave the DNA segments into different length fragments.

15. The method as set forth in claim 11 wherein the DNA fragments are reacted only with reporter labeled dideoxynucleotides.

16. The method as set forth in claim 11 wherein the first restriction enzyme is an ambiguous-end restriction enzyme.

17. The method as set forth in claim 13 wherein the first restriction enzyme is an ambiguous-end restriction enzyme.

18. The method set forth in claim 11 wherein the first restriction enzyme is an exact-end restriction enzyme.

19. The method set forth in claim 13 wherein the first restriction enzyme is an exact-end restriction enzyme.

20. The method as set forth in claim 11 wherein the ratio of labeled dideoxynucleotides to unlabeled deoxynucleotides is varied inversely according to the number of nucleotides in the overhang strands.

21. The method as set forth in claim 13 wherein a third enzyme is used in conjunction with and in addition to the first enzyme or second enzyme to cleave the DNA segments into different length fragments.

22. The method as set forth in claim 13 wherein the DNA fragments are reacted only with reporter labeled dideoxynucleotides.

23. The method as set forth in claim 13 wherein the first restriction enzyme is an ambiguous-end restriction enzyme.

24. A method for mapping DNA segments made up of duplex strands of nucleotides using specific binding pairs, one member of the pair being immobilized on a solid support, by the steps of:

(a) cleaving each segment with a first restriction enzyme to produce fragments of DNA each having a cleaved end,

(b) derivatizing cleaved end nucleotides in each fragment with the other member of the specific binding pair,

(c) cleaving the derivatized DNA fragments with a second restriction enzyme to produce short DNA fragments,

(d) binding the short fragments to the immobilized member, leaving a free end nucleotide on each derivatized fragment,

(e) separating the derivatized fragments from the non-derivatized fragments,

(f) attaching a reporter to one of the free end nucleotides in each derivatized shorter fragment,

(g) separating the reporter labeled, separated fragments from the solid support, and

(h) fractionating the reporter labeled fragments according to size.

25. The method set forth in claim 24 which includes the step of:

analyzing the fractionated fragments for the presence of labeled fragments as a function of size.

26. The method of claim 25 wherein the reporter is nucleotide specific.

27. The method of claim 24 wherein the reporter is nucleotide specific.

28. The method of claim 27 wherein the solid support is beads that are water insoluble and stable to the physical and chemical conditions of steps d, e, f and g of claim 24.

29. The method of claim 28 wherein the beads are chromium dioxide.

30. The method of claim 29 wherein the beads are separated from their surrounding environment by a magnetic field.

31. The method of claim 26 wherein the solid support is beads that are water insoluble and stable to the physical and chemical conditions of steps d, e, f and g of claim 24.

32. The method of claim 31 wherein the beads are chromium dioxide.

33. The method of claim 28 wherein the step of subjecting the short fragments to the immobilized member to attach the derivatized short fragments to the beads includes the step of separating the beads with attached derivatized short fragments from the non-derivatized fragments.

34. The method of claim 33 wherein the beads are chromium dioxide.

35. The method of claim 34 wherein the step of separating the beads is accomplished by the use of a magnetic field.

36. The method of claim 24 wherein the free end nucleotide is on the 3'"'"' strand of DNA.

37. A method for mapping DNA segments made up of duplex strands of nucleotides using specific binding substances (one and the other), one substance being immobilized on a solid support, by the steps of:

cleaving each segment with a first restriction enzyme to produce fragments of DNA each having a cleaved end,

attaching the other substance to the cleaved ends to provide anchor ends,

cleaving the DNA fragments with a second restriction enzyme to provide shorter DNA fragments and free ends,

separating shorter fragments with an anchor end from the remaining shorter fragments by binding the anchor ends to the one substance,

attaching reporter labeled nucleotides to the free end of each separated shorter fragment,

removing the separated shorter fragments from the one substance,

fractionating the removed shorter fragments.

38. A method for mapping DNA segments made up of duplex strands of nucleotides using specific binding pairs, one member of the pair being immobilized on a solid support, by the steps of:

(a) cleaving each segment with a first restriction enzyme to produce fragments of DNA each having a cleaved end,

(b) derivatizing cleaved end nucleotides in each fragment with the other member of the specific binding pair,

(c) cleaving the derivatized DNA fragments with a second restriction enzyme to produce short DNA fragments, the second cleaved ends being non-derivatized,

(d) attaching a reporter to each of the non-derivatized ends of the short fragments,

(e) binding the short fragments with derivatized ends to the one member,

(f) separating the bound, derivatized fragments, some of which are reporter-labeled at one end, from the fragments that are not derivatized at either end,

(g) separating the bound, reporter-labeled fragments from the solid support,

(h) fractionating the reporter labeled fragments according to size.

39. A method for mapping DNA segments made up of duplex strands of nucleotides using specific binding pairs, one member of the pair being immobilized on a solid support, by the steps of:

(a) cleaving each segment with a first restriction enzyme to produce fragments of DNA each having a cleaved end,

(b) derivatizing cleaved end nucleotides in each fragment with the other member of the specific binding pair,

(c) cleaving the derivatized DNA fragments with a second restriction enzyme to produce short DNA fragments, binding the derivatized fragments to the one member,

(d) cleaving the bound DNA fragments with a second restriction enzyme to produce short DNA fragments, some of which have one bound, derivatized and one free end and some of which are not derivatized at either end,

(e) separating the bound short fragments from the unbound short fragments,

(f) attaching a reporter to one of the free end nucleotides in each bound short fragment,

(g) separating the reporter-labeled, bound fragments from the solid support,

(h) fractionating the reporter labeled fragments according to size.