Low sequence bias single-stranded DNA ligation

• US 10,640,828 B2
• Filed: 10/17/2017
• Issued: 05/05/2020
• Est. Priority Date: 01/09/2013
• Status: Active Grant

• Current Assignee: Penn State Research Foundation
• Original Assignee: Penn State Research Foundation
• Inventors: Bevilacqua, Philip C., Assmann, Sarah M., Ding, Yiliang, Kwok, Chun Kit
• Primary Examiner(s): Woolwine, Samuel C
• Application Number:
  US15/785,680
• Publication Number:
  US 20180030494A1
• Time in Patent Office: 931 Days
• Field of Search: None
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Y 605/01001 DNA ligase (ATP) (6.5.1.1)

Abstract

The invention provides compositions and methods for ligating single stranded nucleic acids wherein the ligation is based on fast, efficient, and low-sequence bias hybridization of an acceptor molecule with a donor molecule. In one embodiment, the structure of the donor molecule comprises a stem-loop intramolecular nucleotide base pairing (i.e., hairpin) and a 3′-overhang region such that the overhang is able to hybridize to nucleotides present in the 3′ end of the acceptor molecule.

Claims

1. A composition comprising a donor nucleic acid molecule, wherein the molecule comprises a stem-loop structure and a 3′

overhang, further wherein the molecule comprises a continuous primer binding site that encompasses a portion of the stem and a portion of the loop structure, wherein the donor molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO;

5 through SEQ ID NO;

22, SEQ ID NO;

28 and SEQ ID NO;

29.

2. The composition of claim 1, wherein the stem portion of the stem-loop structure comprises at least 3 nucleotide base pairs and at least one mismatch pair.

3. The composition of claim 1, wherein the 3′

overhang comprises at least 4 nucleotides.

4. The composition of claim 1, wherein the primer binding site comprises 8 nucleotides.

5. The composition of claim 1, wherein the donor molecule is hybridized to a single stranded nucleic acid acceptor molecule to form a hybridized molecule comprising the donor molecule and the acceptor molecule.

6. The composition of claim 5, wherein the hybridized molecule is stable at a temperature as high as 65°

C.

7. A kit comprising a donor nucleic acid molecule, wherein the molecule comprises a stem-loop structure and a 3′

overhang, further wherein the molecule comprises a continuous primer binding site that encompasses a portion of the stem and a portion of the loop structure, wherein the donor molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO;

5 through SEQ ID NO;

22, SEQ ID NO;

28 and SEQ ID NO;

29.

8. The kit of claim 7, further comprising a DNA ligase.

9. The kit of claim 8, wherein the DNA ligase is T4 DNA ligase.