Discriminative analysis of clone signature

• US 20050176007A1
• Filed: 10/15/2002
• Published: 08/11/2005
• Est. Priority Date: 02/11/2002
• Status: Abandoned Application

• Current Assignee: Gemone Express
• Original Assignee: Gemone Express
• Inventors: De Leeuw, Marcel, Issartel, Jean-Paul, Mouret, Jean-Francois
• Application Number:
  US10/503,953
• Publication Number:
  US 20050176007A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators

Abstract

The invention relates to signatures obtained by a method for analyzing nucleic acid, wherein said nucleic acids are partially sequenced, not using all 4 chain extension terminators, a signature is generated, corresponding to the partial sequence, said signature being compared with other theoretical or actual signatures to analyze said nucleic acid.

Claims

1. A reproducible, discriminative and x-coherent signature from a nucleic acid molecule, comprising a signature obtained by a method comprising the steps of:

a) performing an enzymatic chain elongation reaction, using at least one primer to start the elongation process, and at least one chain extension terminator, whereas not all four possible terminators are used, in order to obtain differentially sized products, wherein said differentially sized products emit a detectable signal,

b) separating the differentially sized products obtained in step a),

c) detecting the detectable signals corresponding to each of the differentially sized reaction products after separation, and

d) creating the signature directly or indirectly based on the detected signals in step c).

2. The signature of claim 1, consisting of a signature obtained by a method comprising the steps a) to d) of claim 1.

3. The signature of claim 1 or 2, wherein exactly one chain extension terminator is used.

4. The signature of claim 1 or 2, wherein exactly two chain extension terminators are used.

5. The signature of claim 1 or 2, wherein exactly three chain extension terminators are used.

6. The signature of claim 1, wherein said chain extension terminator is a dideoxyribonucleotide.

7. The signature of claim 1, wherein only one primer is used in said enzymatic chain elongation reaction.

8. The signature of claim 1, wherein more than one primer is used in said enzymatic chain elongation reaction.

9. The signature of claim 8, wherein two primers are used in said enzymatic chain elongation reaction, starting from the 3′

and 5′

ends of said nucleic acid molecule.

10. The signature of claim 1, wherein separation of said differentially sized products is performed by mass spectrometry.

11. The signature of claim 10, wherein said signature in step d) is created from a graph, drawn on the basis of the detected signals in step c).

12. The signature of claim 10, wherein said signature in step d) is created directly from the signals obtained in step c).

13. The signature of claim 12, wherein said signature comprises the intensity of said signals and/or the difference of mass between two consecutive signals.

14. The signature of claim 1, wherein separation of said differentially sized products is performed by denaturing electrophoresis.

15. The signature of claim 14, wherein said signature in step d) is created from or is an electropherogram, based on the detected signals in step c).

16. The signature of any one of claims 14 and 15, wherein said signature in step d) is created directly from the signals obtained in step c).

17. The signature of claim 16, wherein said signature comprises the intensity of said signals and/or the time lapse or fragment length in bases between two consecutive signals.

18. The signature of claim 15, wherein said electropherogram is obtained from an ABI 3700 DNA Sequencer (Perkin Elmer, Applied BioSystems, Inc).

19. The signature of claim 15, wherein said electropherogram is created with means of a computer-assisted “

basecalling”

.

20. The signature of claim 19, wherein said computer-assisted “

basecalling”

is performed with the Sequencher software (Gene Codes Corporation).

21. The signature of claim 1, wherein said enzymatic chain elongation reaction of step a) is performed on a limited number of bases of said nucleic acid molecule, for example about 200 bases.

22. The signature of claim 1, wherein said detectable signal is carried by a label on said primer.

23. The signature of claim 1, wherein said detectable signal is carried by a label on said chain extension terminator.

24. The signature of any one of claims 22 and 23, wherein said label is a fluorescent label.

25. The signature of any one of claims 22 and 23, wherein said label is a radioactive label.

26. The signature of claim 14, wherein said electrophoresis is a capillary electrophoresis.

27. The signature of claim 14, wherein said electrophoresis and signal detection steps is simultaneously performed on multiple nucleic acid molecules subjected to step a), wherein said detectable signal emitted by the chain elongation products is different for each of the multiple nucleic acid molecules.

28. The signature of claim 27, wherein said detection step comprises the step of detecting, distinguishing (demultiplexing) and recording the signals specific to each of the multiple nucleic acid molecules.

29. The signature of claim 28, wherein said demultiplexing is computer- assisted.

30. The signature of claim 1, wherein said enzymatic chain elongation reaction is performed with thermosequenase.

31. The signature of claim 1, wherein said enzymatic chain elongation reaction is performed with TAQ polymerase.

32. The signature of claim 1, consisting of a string of alphanumeric characters with a specific character for the bases corresponding to specific nucleotide identified due to the presence of the used chain extension terminators, and a “

joker”

sign for the other bases, wherein said method further comprises the step of allocating the alphanumeric character (s) corresponding to the bases detected by the used terminator (s), and a joker sign corresponding to the non detected bases.

33. The signature of claim 32, wherein exactly one chain extension terminator is used.

35. A method for analyzing the differences of gene expression between at least two samples, comprising the steps of:

a) for each of the nucleic acids present in said at least two samples, obtaining a signature in accordance with the method of claim 1,

b) determining the number of occurrences of a given signature in each sample,

c) comparing the number of occurrences of said given signature between said samples, and

d) relating the difference observed in the level of occurrences said given signature to the differential expression of the genes leading to said signature, between said at least two samples.

36. The method of claim 35, including a step of normalization of the number of occurrences of the signatures versus an internal standard (such as actin).

37. The method of claim 35, wherein said samples are cDNA libraries obtained from mRNA from two samples (cells, tissues . . . ) submitted to different conditions.

38. The method of claim 37, wherein said different conditions are selected from the group consisting of sick/healthy, tumoral/non tumoral, difference of stress, and difference of tissues.

39. A method for sequencing a large DNA, comprising:

a) performing a random shotgun sequencing method on said DNA, fragmented and cloned within a library,

b) for a clone in the library, obtaining a signature in accordance with the method of claim 1,

c) comparing said signature for said clone to the theoretical or genuine signatures of the contigs assemblies in progress, to determine if said signature for said clone is fully represented within said theoretical signatures,

d) sequencing said clone if the answer obtained in step c) is negative, and

e) starting the method over from step b) on another clone in the library if the answer in step c) is positive.

40. The method of claim 39, wherein said large DNA is a genome, in particular a bacterial, eukaryotic or chromosomal genome, or a plasmid or an organelle genome.

41. A method for identifying genomic differences between a first organism, the genomic sequence of which is known, and a second organism, the genomic sequence of which is unknown, comprising:

a) fragmenting and cloning genomic DNA of said second organism in a library,

b) for a clone in the library, obtaining a signature in accordance with method of claim 1,

c) comparing said signature for said clone to the theoretical or genuine genomic signature of said first organism, to determine if said signature for said clone is fully represented within said theoretical signature,

d) deducing the presence of a difference between said second organism and said first organism, when said signature for said clone is not fully represented within said theoretical signature, and optionally, and

e) sequencing said clone to characterize said difference.

42. A method for analyzing the expressed genes from a cell type or a tissue, from a cDNA library obtained from total mRNA from said cell type or tissue, comprising the steps of:

a) spotting the clones of said cDNA library on a solid support,

b) selecting a random subset of clones in said cDNA library,

c) on each clone on said random subset of step b), obtaining a signature in accordance with the method of claim 1,

d) comparing said signatures and clustering the clones according to the similarities between said signatures obtained in step c),

e) choosing and labeling the cDNA carried by the clones which are highly represented in said subset, (representation more than 2%),

f) hybridizing said labeled cDNA to said solid support,

g) creating a cDNA sub-library consisting of the clones for which no hybridization has been observed in stepf), and

h) repeating said steps b) to g) on said sub-library as long as the number of clones in said cDNA sub-library remains too high.

43. A method for creating a normalized cDNA library from a cell type or a tissue, comprising the steps of performing the method of claim 42, in order to identify the total mRNA present in said cell type or tissue, and creating said normalized library, by clustering the clones representing all expressed genes, and optionally indicating their proportion in said cell type or tissue.

44. A normalized library obtained by the method of claim 43.

45. A method for designing nucleic acid arrays bearing probes complementary to genes that are expressed at a similar level in a cell type or tissue, comprising the steps of:

performing the method of claim 42, wherein said labeled cDNA at each step e) represent genes that are expressed at a similar level in said cell type or tissue,

selecting probes complementary to said labeled cDNA in each steps, and

designing said nucleic acid array by fixing said probes to a solid surface.

46. A nucleic acid array obtained by the method of claim 45.

34. A signature specific for a nucleic acid molecule, consisting of an electropherogram obtained after electrophoresis of the product of an enzymatic chain elongation reaction on said nucleic acid molecule, using at least one primer to start the elongation process, and at least one chain extension terminator, whereas not all four possible terminators are used.

47. A method for generating graphical data representative of the similarity of first and second x-coherent analog signatures of nucleic acid molecules, comprising the steps of:

dividing each signature into a plurality of fragments,

performing a cross-correlation of each fragment of the first signature with each fragment of the second signature, respectively, so as to generate a matrix of cross-correlation values,

generating a matrix of graphical zones for display, wherein each zone has a visual property determined from a corresponding cross- correlation value, and

displaying said matrix.

48. The method of claim 47, wherein the fragments are overlapping.

49. The method of claim 47, wherein each signal comprises individual peaks, wherein each fragment contains a plurality of peaks, preferably from 10 to 20 peaks.

50. The method of claim 47, wherein each visual property is selected from a color or a grayscale level.

51. The method of claim 50, wherein a clearer color or grayscale value corresponds to a higher correlation between fragments.