Method for assembly of polynucleic acid sequences

• US 8,999,679 B2
• Filed: 12/18/2009
• Issued: 04/07/2015
• Est. Priority Date: 12/18/2008
• Status: Active Grant

• Current Assignee: ITI Scotland Limited (Scottish Government)
• Original Assignee: ITI Scotland Limited (Scottish Government)
• Inventors: Che, Austin, Knight, Tom, Canton, Barry, Kelly, Jason, Shetty, Reshma
• Primary Examiner(s): Ketter, Jim
• Application Number:
  US13/140,473
• Publication Number:
  US 20120040870A1
• Time in Patent Office: 1,936 Days
• Field of Search: None
• US Class Current: 435/91.41
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• 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

Abstract

The present invention provides a method for the assembly of a polynucleic acid sequence from a plurality of nucleic acid sequences in which the polynucleic acid sequence is of a formula N_n+1, in which N represents a nucleic acid sequence and where n is 1 or greater than 1 and each N may be the same or a different nucleic acid sequence, in which the method comprises: (i) providing a first nucleic acid sequence N1 which has an oligonucleotide linker sequence L1³ at the 3′-end of the nucleic acid sequence; (ii) providing a second nucleic acid sequence N2 which optionally has an oligonucleotide linker sequence L2^3′ at the 3′-end of the nucleic acid sequence and which has an oligonucleotide linker sequence L2^5′ at the 5′-end of the nucleic acid sequence, wherein the 5′-end linker sequence L2^5′ of nucleic acid sequence N2 is complementary to the 3′-end linker sequence L1^3′ of nucleic acid sequence N1; (iii) optionally providing one or more additional nucleic acid sequences N, wherein nucleic acid sequence N2 has an oligonucleotide linker sequence L2^3′ at the 3′-end of the nucleic acid sequence, and wherein said one or more additional nucleic acid sequences N comprises a terminal additional nucleic acid sequence NZ, and wherein each additional nucleic acid sequence N has an oligonucleotide linker sequence at its 3′-end, wherein said terminal additional nucleic acid sequence NZ optionally lacks an oligonucleotide linker sequence at its 3′-end and wherein each additional nucleic acid sequence N has an oligonucleotide linker sequence at its 5′-end, wherein for the first additional nucleic acid sequence N3 the 5′-end linker sequence L3^5′ is complementary to the 3′-end linker sequence L2^3′ of nucleic acid sequence N2 and for each second and subsequent additional nucleic acid sequence N the 5′-end linker sequence is complementary to the 3′-end linker sequence of the respective preceding additional nucleic acid sequence; and (iv) ligating said nucleic acid sequences to form said polynucleic acid sequence.

Claims

1. A method for the assembly of a polynucleic acid sequence from a plurality of nucleic acid sequences in which the polynucleic acid sequence is of a formula N_n+1, in which N represents a nucleic acid sequence and where n is 1 or greater than 1 and each N may be the same or a different nucleic acid sequence, in which the method comprises:

(i) providing a first nucleic acid sequence N1 in a vector, using a restriction enzyme to cut the nucleic acid sequence out from the vector and ligating an oligonucleotide linker sequence L1^3′

to the 3′

-end of the nucleic acid sequence;

(ii) providing a second nucleic acid sequence N2 in a vector, using a restriction enzyme to cut the nucleic acid sequence out from the vector, optionally ligating an oligonucleotide linker sequence L2^3′

to the 3′

-end of the nucleic acid sequence and ligating an oligonucleotide linker sequence L2^5′

to the 5′

-end of the nucleic acid sequence, wherein the 5′

-end linker sequence L2^5′

of nucleic acid sequence N2 is complementary to the 3′

-end linker sequence L1^3′

of nucleic acid sequence N1;

(iii) optionally providing one or more additional nucleic acid sequence(s) N in one or more vector(s), using a restriction enzyme to cut the nucleic acid sequence(s) out from the vector(s), wherein nucleic acid sequence N2 has an oligonucleotide linker sequence L2^3′

at the 3′

-end of the nucleic acid sequence, and wherein said one or more additional nucleic acid sequences N comprises a terminal additional nucleic acid sequence NZ, and ligating an oligonucleotide linker sequence to the 3′

-end of each additional nucleic acid sequence N, wherein said terminal additional nucleic acid sequence NZ optionally lacks an oligonucleotide linker sequence at its 3′

-end, and ligating an oligonucleotide linker sequence to the 5′

-end of each additional nucleic acid sequence N, wherein for the first additional nucleic acid sequence N3 the 5′

-end linker sequence L3^5′

is complementary to the 3′

-end linker sequence L2^3′

of nucleic acid sequence N2 and for each second and subsequent additional nucleic acid sequence N the 5′

-end linker sequence is complementary to the 3′

-end linker sequence of the respective preceding additional nucleic acid sequence; and

(iv) ligating said nucleic acid sequences to form said polynucleic acid sequence.

2. A method according to claim 1, wherein said first nucleic acid sequence N1 has an oligonucleotide linker sequence L1^5′

at the 5′

-end of the nucleic acid sequence.

3. A method according to claim 1, wherein said second nucleic acid sequence N2 has an oligonucleotide linker sequence L2^3′

at the 3′

-end of the nucleic acid sequence.

4. A method according to claim 1, wherein said terminal additional nucleic acid sequence NZ has an oligonucleotide linker sequence LZ^3′

at the 3′

-end of the nucleic acid sequence.

5. A method according to claim 3, wherein said first nucleic acid sequence N1 has an oligonucleotide linker sequence L1^5′

at the 5′

-end of the nucleic acid sequence;

wherein said second nucleic acid sequence N2 has an oligonucleotide linker sequence L2^3′

at the 3′

-end of the nucleic acid sequence; and

wherein the 5′

-end linker sequence L1^5′

of nucleic acid sequence N1 is complementary to the 3′

-end linker sequence L2^3′

of nucleic acid sequence N2.

6. A method according to claim 4, wherein said first nucleic acid sequence N1 has an oligonucleotide linker sequence L1^5′

at the 5′

-end of the nucleic acid sequence;

wherein said terminal additional nucleic acid sequence NZ has an oligonucleotide linker sequence LZ^3′

at the 3′

-end of the nucleic acid sequence; and

wherein the 5′

-end linker sequence L1^5′

of nucleic acid sequence N1 is complementary to the 3′

-end linker sequence LZ^3′

of nucleic acid sequence NZ.

7. A method according to claim 1, wherein each of said 3′

-end linker sequences and said 5′

-end linker sequences is partially double stranded.

8. A method according to claim 1, wherein each said nucleic acid sequence has an overhang at each end.

9. A method according to claim 8, wherein said overhang at each end of said nucleic acid sequence is produced by digestion with one or more restriction enzymes.

10. A method according to claim 8, wherein said overhang is 3 or 4 nucleotides in length.

11. A method according to claim 8, wherein the overhang at the 3′

-end of the nucleic acid sequence and/or at the 5′

-end of the nucleic acid sequence is the same for each nucleic acid sequence.

12. A method according to claim 1, wherein said nucleic acid sequences are purified immediately prior to step (iv).

13. A method according to claim 12, wherein said nucleic acid sequences are purified using a method selected from the group consisting of DNA purification spin columns, gel extraction and biotin-based purification.

14. A method according to claim 1, wherein each said nucleic acid sequence is a protein coding sequence or a regulatory or control element.

15. A method according to claim 1, wherein step (iv) is carried out using DNA ligase.

16. A method for the preparation of a library of polynucleic acid sequences, the method comprising simultaneously producing a plurality of different polynucleic acid sequences using the method of claim 1.