Method for constructing an oligonucleotide concatamer library by rolling circle replication

• US 5,648,245 A
• Filed: 05/09/1995
• Issued: 07/15/1997
• Est. Priority Date: 05/09/1995
• Status: Expired due to Term

• Current Assignee: Carnegie Institution of Washington
• Original Assignee: Carnegie Institution of Washington
• Inventors: Xu, Si-Qun, Fire, Andrew
• Primary Examiner(s): Guzo, David
• Assistant Examiner(s): Wai, Thanda
• Application Number:
  US08/437,538
• Time in Patent Office: 798 Days
• Field of Search: 435/91.1, 435/91.4, 435/91.41
• US Class Current: 435/91.1
• 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: C12Q 1/6844 Nucleic acid amplification reactions

Abstract

An in vitro method for constructing an oligonucleotide concatamer library is revealed. The method has four steps. The first step involves the generation of a primer-bridged circular template oligonucleotide. In the second step, the primer-bridged circular template is used to generate a single-stranded oligonucleotide concatamer by rolling circle replication. In the third step, the single-stranded oligonucleotide concatamer is converted to a double-stranded oligonucleotide concatamer. Finally, the double-stranded oligonucleotide concatamer is cloned or used directly in in vitro assays which allow molecules of interest to be isolated or amplified.

Claims

1. An in vitro method for constructing an oligonucleotide concatamer library, comprising the steps of:

a) generating a population of primer-bridged circular template oligonucleotides by annealing linear primer oligonucleotides to linear template oligonucleotides and ligating said linear template oligonucleotides into circular template oligonucleotides;

b) generating a population of single-stranded oligonucleotide concatamers by rolling circle replication of said primer-bridged circular template oligonucleotides; and

c) generating a population of corresponding double-stranded oligonucleotide concatamers by synthesizing single stranded oligonucleotides which are complementary to said population of single-stranded oligonucleotide concatamers.

2. The method of claim 1, further comprising the step of:

d) cloning said double-stranded oligonucleotide concatamers.

3. The method of claim 1, wherein said primer-bridged circular template oligonucleotides are generated by:

i) producing linear single-stranded template oligonucleotides having a phosphorylated 5'"'"' end and a single-stranded primer oligonucleotide with permuted complementarity to said linear single-stranded template oligonucleotides;

ii) annealing said linear single-stranded primer oligonucleotides to said linear single-stranded template oligonucleotides; and

iii) ligating said linear single-stranded template oligonucleotides to form primer-bridged circular template oligonucleotides.

4. The method of claim 1, wherein said population of single-stranded oligonucleotide concatamers is generated by reacting said primer-bridged circular template oligonucleotides with nucleic acid polymerase in the presence of nucleoside triphosphates to form single-stranded oligonucleotide concatamers.

5. The method of claim 1, wherein said population of single-stranded oligonucleotide concatamers is converted to a population of double-stranded oligonucleotide concatamers by reacting said population of single-stranded oligonucleotide concatamers with nucleic acid polymerase in the presence of nucleoside triphosphates.

6. The method of claim 2, wherein said population of double-stranded oligonucleotide concatamers is cloned by:

i) inserting said double-stranded oligonucleotide concatamers into a plasmid vector to obtain a mixture of ligation products; and

ii) transforming a host cell with said mixture.

7. The method of claim 6, wherein said host cell is a bacterial cell.

8. The method of claim 1, wherein said oligonucleotide concatamer library is a DNA oligonucleotide concatamer library.

9. The method of claim 4, wherein said nucleic acid polymerase is a DNA polymerase.