Use of the combing process for the indentification of DNA origins of replication

• US 20050142593A1
• Filed: 11/05/2004
• Published: 06/30/2005
• Est. Priority Date: 05/28/1999
• Status: Abandoned Application

• Current Assignee: Institut Pasteur and Centre National de La Recherche Scientifique
• Original Assignee: Institut Pasteur and Centre National de La Recherche Scientifique
• Inventors: Hyrien, Olivier, Bensimon, Aaron, Herrick, John
• Application Number:
  US10/981,641
• Publication Number:
  US 20050142593A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels

Abstract

Eukaryotic genomes are duplicated by the activation of multiple bidirectional origins of replication. The replication programs of these cells depend on the temporal and spatial organisation of replication origins throughout the genome. To investigate the replication program in a higher eukaryote, we employed a technique called molecular combing. This technique allows for a quantitative analysis of DNA replication on a genome wide basis. As a model system. Xenopus Laevis sperm chromatin were differentially labelled at successive time points after the beginning of DNA synthesis. Genomic DNA was then extracted and combed on a glass surface. Direct measurements made on the labelled DNA provided a comprehensive analysis of the spatial and temporal organisation of the X. leavis early embryo replication program and revealed that the number of replication origins activated per kilobase increases throughout the period of DNA synthesis.

Claims

1-28. -28. (canceled)

29. A kit for detecting an origin of replication in a DNA sample comprising:

(A) reagents for replicating the DNA in the sample;

(B) labeled nucleotides for incorporation into a region that is an origin of replication of the replicating DNA in the sample of (A);

(C) a surface on which the replicated DNA can be aligned by molecular combing; and

(D) reagents for detecting the labeled nucleotides that are incorporated into the DNA sample of (A) during replication.

30. The kit as claimed in claim 29, further comprising a labeled probe that binds to the labeled nucleotides of (B).

31. The kit as claimed in claim 30, wherein the labeled probe is an antibody conjugated to a label.

32. The kit as claimed in claim 31, wherein the label conjugated to the antibody is fluorescent.

33. The kit as claimed in claim 29, further comprising reagents to wash the DNA following replication of the DNA in the sample in (A).

34. The kit as claimed in claim 29, wherein the sample DNA comprises genomic DNA.

35. The kit as claimed in claim 29, wherein the surface in (C) comprises glass.

36. The kit as claimed in claim 29, wherein the kit further comprises a second labeled nucleotide that is distinguishable from the labeled nucleotide in (B) (the first labeled nucleotide).

37. The kit as claimed in claim 36, wherein the kit further comprises a first labeled probe that detects the first labeled nucleotide and a second labeled probe that detects the second labeled nucleotide.

38. The kit as claimed in claim 37, wherein the labeled probe that detects the first labeled nucleotide is a first antibody conjugated to a first label and the labeled probe that detects the second labeled nucleotide is a second antibody conjugated to a second label, wherein the first and the second labels are different.

39. The kit as claimed in claim 38, wherein the first and the second labels are fluorescent.

40. The kit as claimed in claim 36, wherein the first labeled nucleotide is biotin-dUTP and the second labeled nucleotide is digoxigenin-dUTP.

41. The kit as claimed in claim 29, further comprising a means for measuring the distances between the labeled nucleotides that are incorporated into the sample DNA during replication and the nucleotide probe to determine the location of the origin of DNA replication with respect to the nucleotide probe.

42. The kit as claimed in claim 41, further comprising a probe for detecting the labeled nucleotides.

43. The kit as claimed in claim 42, wherein the probe is an antibody conjugated to a label.

44. The kit as claimed in claim 43, wherein the label conjugated to the antibody is fluorescent.

45. The kit as claimed in claim 41, further comprising reagents to wash the DNA following incubation of the DNA sample in (A).

46. The kit as claimed in claim 41, wherein the sample DNA is genomic DNA.

47. The kit as claimed in claim 41, wherein the surface in (C) is glass.