Error-free sequencing of DNA

• US 10,273,538 B2
• Filed: 02/05/2015
• Issued: 04/30/2019
• Est. Priority Date: 02/05/2014
• Status: Active Grant

• Current Assignee: Fraunhofer Gesellschaft Zur Foerderung Der Angewandten Forsching E.V.
• Original Assignee: Fraunhofer Gesellschaft Zur Foerderung Der Angewandten Forsching E.V.
• Inventors: Klein, Christoph, Kirsch, Stefan, Czyz, Zbigniew Tadeusz, Lahrmann, Urs
• Primary Examiner(s): Kim, Young J
• Application Number:
  US15/116,657
• Publication Number:
  US 20160348164A1
• Time in Patent Office: 1,545 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences

Abstract

The invention relates to a novel method of error-free sequencing of DNA. Further, the present invention provides for a four-part oligonucleotide, comprising a fixed sequence, a randomized sequence, a restriction nuclease recognition site and/or restriction site, and a primer binding site. The invention also relates to the use of the sequenced DNA fragments obtained by the methods of the invention in methods for DNA sequence analysis, generation of cell lineage trees or assessment of copy numbers.

Claims

1. A method of error-free sequencing of DNA, comprising the steps of:

(a) providing a sample comprising DNA;

(b) digesting the DNA with a restriction endonuclease under conditions suitable to obtain DNA fragments of similar length,

wherein said restriction endonuclease is capable of providing 5′

overhangs, wherein the terminal nucleotide of the overhang is phosphorylated or,

wherein said restriction endonuclease is capable of providing 3′

overhangs, wherein the terminal nucleotide of the overhang is hydroxylated on said DNA fragments;

(c) annealing a first oligonucleotide to said DNA fragments, wherein a first sequence of said first oligonucleotide is complementary to the 5′

or 3′

overhang, respectively, of said DNA fragment, and a second sequence of said first oligonucleotide is complementary to a first sequence of a second oligonucleotide, wherein said second oligonucleotide comprises a second and a third sequence, wherein said second sequence of said second oligonucleotide comprises a randomized sequence;

(d) ligating said second oligonucleotide to said DNA fragment;

(e) filling in of the generated overhangs;

(f) amplifying said DNA fragments using a third oligonucleotide comprising a sequence binding to said third sequence of said second oligonucleotide; and

(g) sequencing said amplified DNA fragments.

2. The method of claim 1, wherein said second oligonucleotide further comprises a fourth sequence comprising a restriction site of a site-specific endonuclease.

3. The method of claim 2, wherein said method further comprises the step (f′

) wherein a homing endonuclease is added in said step (f′

).

4. The method of claim 1, wherein said second oligonucleotide is a DNA oligonucleotide, an RNA oligonucleotide or a DNA/RNA oligonucleotide.

5. The method of claim 1, wherein said method further comprises the step (e′

), wherein an exonuclease is added in said step (e′

).

6. The method of claim 5, wherein said exonuclease is an enzyme degrading single-stranded DNA, RNA or DNA/RNA molecules.

7. The method of claim 1, wherein said DNA comprises (i) the genome or transcriptome of a single cell, (ii) chromosome(s) of a single cell, (iii) nucleic acids from exosomes or other microvesicles of a single cell or (iv) fragment(s) or subfraction(s) of the material of any one of items (i) to (iii).

8. The method of claim 1, wherein said DNA comprises (i) the DNA of more than one single cell, (ii) cell-free fetal DNA of more than one single cell, (iii) cell-free DNA of more than one single cell in serum and/or plasma of cancer patients or (iv) fragment(s) or subfraction(s) of the material of any one of items (i) to (iii).

9. The method of claim 1, wherein said restriction endonuclease is MseI or an isoschizomer thereof.

10. The method of claim 1, wherein said randomized sequence comprises 3 to 24 nucleotides.

11. The method of claim 1, wherein said first oligonucleotide has the sequence 5′

-TAACTGACdd-3′ and

/or wherein said second oligonucleotide has the sequence as shown in SEQ ID NO;

1 and/or wherein said third oligonucleotide has the sequence as shown in SEQ ID NO;

2.

12. The method of claim 1, wherein the last 3′

nucleotide of the first oligonucleotide is a dd-nucleotide.