DNA-ADAPTER-MOLECULES FOR THE PREPARATION OF DNA-LIBRARIES AND METHOD FOR PRODUCING THEM AND USE

• US 20160289755A1
• Filed: 09/29/2014
• Published: 10/06/2016
• Est. Priority Date: 09/30/2013
• Status: Abandoned Application

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Hahn, Peter, Azzawi, Alexander, Grnefeld, Peter
• Application Number:
  US15/025,787
• Publication Number:
  US 20160289755A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety

Abstract

The invention relates to DNA-adapter-molecules for the preparation of DNA-libraries and methods for producing them and their use. The invention is useful for the application in molecular biology, in particular for Next Generation Sequencing and/or Library Multiplexing. The present invention discloses DNA-adapter-molecules, comprising a double-stranded polynucleotide molecule, whereat the 5′ end of the first strand is modified in a way, that no binding site for kinases is available, the 3′ end of the first strand is modified in a way that no ligation can occur, the 5′ end of the reverse strand is modified in a way, that no binding site for a kinase is available, and the 3′ end of the reverse strand features a free hydroxyl group (at the 3′ position of the last nucleotide).

Claims

1-15. -15. (canceled)

16. A DNA-adapter-molecule comprising a double-stranded molecule, wherein the double-stranded molecule comprises a first strand and a reverse strand,

a. wherein first nucleotide at the 5′

end of the first strand contains a modified hydroxyl group and does not contain a free phosphate;

b. wherein the last nucleotide at the 3′

end of the first strand does not contain a free hydroxyl group;

c. wherein first nucleotide at the 5′

end of the reverse strand contains a modified hydroxyl group and does not contain a free phosphate; and

d. wherein the 3′

end of the reverse strand contains a free hydroxyl group.

17. The DNA-adapter-molecule of claim 16, further comprising a barcode sequence and a binding site for amplification and sequencing primers.

18. The DNA-adapter-molecule of claim 16, wherein the DNA-adapter-molecule comprises 20 to 90, 40 to 70, or 40 to 60 base pairs.

19. The DNA-adapter-molecule of claim 16, wherein the modified hydroxyl group at the 5′

end of the first strand and the reverse strand is esterified or etherified.

20. The DNA-adapter-molecule of claim 16, wherein the 5′

end of the reverse strand comprises a 5′

-C3-spacer or 5′

-D-spacer.

21. The DNA-adapter-molecule of claim 16, wherein the first strand further comprises a C3-spacer or an O-methyl deoxynucleotide at the 5′

end.

22. The DNA-adapter-molecule of claim 16, wherein the 3′

end of the first strand comprises a C3-spacer or an amino-modifier.

23. A method of producing a DNA-adapter-molecule, wherein the DNA-adapter-molecule comprises a first strand and a reverse strand, said method comprising:

a. modifying the 5′

end of the first strand, wherein said modification results in a modified hydroxyl group and that results in the absence of a free phosphate,

b. modifying the 3′

end of the first strand, wherein said modification results in a modified hydroxyl group, and

c. modifying the 5′

end of a reverse strand wherein said modification results in a modified hydroxyl group and that results in the absence of a free phosphate, thereby producing a DNA-adapter-molecule.

24. The method of claim 23, wherein the modification of the 5′

end of the first strand in step a) comprises attaching a 5′

-O-methyl deoxythymidine monophosphate or a 5′

-C3-spacer to the 5′

end of the first strand.

25. The method of claim 23 wherein the modification of the 3′

end of the first strand step b) comprises attaching a 3′

-C3-spacer or 3′

-amino-modifiers to the 3′

end of the first strand.

26. The method of claim 23, wherein the modification of the 5′

end of the reverse strand step c) comprises attaching a 5′

-C3-spacer or 5′

-D-spacer to the 5′

end of the reverse strand.

31. A kit comprising:

a. one or more DNA-adapter-molecules, and b. a DNA polymerase with 3′

to 5′

exonuclease activity and 5′

to 3′

polymerase activity, deoxynucleoside triphosphates, a polynucleotide kinase, a ligase, a DNA polymerase with 3′

to 5′

exonuclease activity, or a buffer.