Method, compositions and kits for preparation of nucleic acids

Abstract

Methods, compositions and kits are provided for labeling, copying and/or amplifying nucleic acids. The methods, compositions and kit can be used for a variety of applications, for example, genome-wide scanning applications such as CGH or location analysis.

Description

Claims

1. A method comprising: contacting a sample of non-bacteriophage, non-circular, genomic DNA with a T7-like polymerase in the presence of at least one accessory protein, an oligonucleotide capable of binding to a sequence of the non-bacteriophage genomic DNA, and one or more nucleotides, under conditions wherein the oligonucleotide binds to the sequence of the non-bacteriophage genomic DNA and the T7-like polymerase extends the primer.

2. The method of claim 1, wherein the at least one accessory protein is selected from the group consisting of a thioredoxin, a helicase, a primase, a single-stranded binding protein, functionally equivalent proteins, and combinations thereof.

3. The method of claim 1, wherein the at least one accessory protein is obtained by overexpressing a recombinant form of the protein in a host cell.

4. The method of claim 1, wherein the method further comprises reconstituting a T7-like DNA polymerase holoenzyme in vitro.

5. The method of claim 1, wherein contacting is done in the presence of a thioredoxin, a helicase, a primase, and a single-stranded binding protein.

6. The method of claim 2, wherein helicase and primase activities are provided in a single protein.

7. The method of claim 1, wherein the sample of genomic DNA has the complexity of at least an E. coli genome.

8. The method of claim 1, wherein the sample of genomic DNA has the complexity of a mammalian genome.

9. The method of claim 1, wherein contacting occurs in the presence of a plurality of random or degenerate sequence oligonucleotides.

10. The method of claim 1, wherein at least one of the one or more nucleotides is labeled.

11. The method of claim 1, wherein the contacting occurs under conditions suitable for copying the genomic DNA in the sample.

12. The method of claim 1, wherein the contacting occurs under conditions suitable for labeling the genomic DNA in the sample.

13. The method of claim 12, wherein the contacting occurs under conditions suitable for labeling copied genomic DNA.

14. The method of claim 1, further comprising the step of fragmenting the genomic DNA.

15. The method of claim 14, wherein the fragmenting is performed by contacting the genomic DNA with a nuclease.

16. The method of claim 1, further comprising the step of contacting primer extension products to an array.

17. The method of claim 1, further comprising performing said method on first and second separate samples and mixing primer extension products.

18. The method of claim 17, wherein the primer extension products from the first and second samples are differentially labeled.

19. The method of claim 17, comprising determining relative amounts of at least one sequence in the first and second samples.

20. The method of claim 1, further comprising performing said method on first and second separate samples and contacting primer extension products to the same array or to at least two arrays comprising at least a subset of identical sequences at features of the arrays.

21. The method of claim 1, wherein the genomic sample comprises DNA binding proteins bound thereon and wherein the method comprises a fragmentation step to fragment the genomic DNA at sequences not bound by the DNA binding proteins.

22. The method of claim 21, wherein the DNA binding proteins are crosslinked to the genomic DNA.

23. The method of claim 21, further comprising obtaining DNA fragments bound to a DNA binding protein of interest prior to the contacting step.

24. The method of claim 23, wherein the obtaining comprises an immunoprecipitation step.

25. The method of claim 23, wherein the method further comprises obtaining primer extension products and contacting the products to an array.

26. A method comprising: contacting a test sample and a reference sample of genomic DNA with a T7-like polymerase in the presence of at least one accessory protein, an oligonucleotide primer capable of binding to a sequence of the genomic DNA, and one or more nucleotides, under conditions wherein the oligonucleotide primer binds to the sequence of the genomic DNA in the test and reference samples and the T7-like polymerase extends the primer,obtaining primer extension products from the first and second samples and contacting primer extension products to the same array or to at least two arrays comprising at least a subset of identical sequences at features of the arrays.

27. The method of claim 26, further comprising determining relative amounts of at least one sequence in the test and reference sample.

28. A method comprising: contacting a sample of genomic DNA that comprises DNA binding proteins bound thereon;fragmenting the genomic DNA at sequences not bound by the DNA binding proteins;obtaining DNA fragments bound to a DNA binding protein of interest;removing the DNA binding protein of interest from the DNA fragments;contacting the DNA fragments with a T7-like polymerase in the presence of at least one accessory protein, oligonucleotide primers capable of binding to a sequence of a plurality of the fragments, and one or more nucleotides, under conditions wherein the oligonucleotides binds to the sequence of the fragments and T7-like polymerase extends the primer, and contacting primer extension products to an array of nucleic acids.

29. The method of claim 28, further comprising determining the location and/or sequence of a fragment to which the DNA binding protein of interest binds.

30. A kit comprising a T7-like polymerase, at least one accessory protein, and a sample of non-bacteriophage, non-circular genomic DNA.

31. The kit of claim 30, wherein the sample comprises genomic DNA having at least the complexity of E. coli DNA.

32. The kit of claim 30, wherein the sample comprises genomic DNA having at least the complexity of mammalian DNA.

33. A kit comprising a T7-like polymerase, at least one accessory protein, and random or degenerate sequence oligonucleotides for binding to a plurality of genomic DNA sequences, and nucleotides labeled with spectrally distinguishable labels.

34. A kit comprising a T7-like polymerase, at least one accessory protein, and a deparaffinizing reagent.

35. A kit comprising a comprising a T7-like polymerase, at least one accessory protein, and an antigen-binding molecule specific to a DNA binding protein.