Methods and compositions for isolating template nucleic acids

• US 9,267,168 B2
• Filed: 06/11/2013
• Issued: 02/23/2016
• Est. Priority Date: 06/12/2012
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Miller, Erik
• Primary Examiner(s): Mummert, Stephanie K
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US13/915,366
• Publication Number:
  US 20130330722A1
• Time in Patent Office: 987 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• 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 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/162 Helper probe

Abstract

The present invention is directed to methods and compositions for isolating template nucleic acids containing target sequences of interest, wherein those isolated template nucleic acids can be further assessed for information related to sequence and nucleic acid modifications.

Claims

1. A method for isolating circular template nucleic acids containing one or more target sequences from a mixture of nucleic acids, said method comprising:

(a) providing a population of nucleic acid constructs, said constructs comprising circular template nucleic acids, wherein a plurality of said circular template nucleic acids comprises said one or more target sequences;

(b) amplifying said population of nucleic acid constructs with a strand displacing polymerase to produce amplified template nucleic acids, wherein said amplified template nucleic acids each comprise one of said circular template nucleic acids hybridized to a linear nascent strand comprising at least one copy of a sequence complementary to said one or more target sequences;

(c) associating one or more affinity handles with said amplified template nucleic acids, wherein said one or more affinity handles comprises an affinity oligonucleotide moiety and a retrieval moiety, wherein said affinity oligonucleotide moiety hybridizes to said at least one copy of a sequence complementary to said one or more target sequences;

(d) separating amplified template nucleic acids associated with said affinity handles from amplified template nucleic acids not associated with said affinity handles using said retrieval moieties;

(e) after said separating, treating said amplified template nucleic acids such that said circular template nucleic acids are separated from said linear nascent strands; and

(f) isolating said circular template nucleic acids containing one or more target sequences from the mixture of nucleic acids.

2. The method of claim 1, wherein said population of nucleic acid constructs comprises genomic DNA.

3. The method of claim 1, wherein said circular template nucleic acids comprise a single-stranded portion and a double-stranded portion.

4. The method of claim 1, wherein said nucleic acid constructs are single stranded or double stranded.

5. The method of claim 1, wherein said nucleic acid constructs comprise:

(a) a first strand segment,

(b) a second strand segment substantially complementary to said first strand segment;

(c) a first linking oligonucleotide segment joining the 3′

end of said first strand segment to the 5′

end of said second strand segment;

(d) a second linking oligonucleotide segment joining the 5′

end of said first strand segment to the 3′

end of said second strand segment.

6. The method of claim 1, wherein said affinity handles comprises an oligonucleotide or a protein capable of binding to a nucleic acid.

7. The method of claim 1, wherein said affinity handles comprise an oligonucleotide.

8. The method of claim 7, wherein said oligonucleotide comprises deoxynucleotide bases, ribonucleotide bases, or a combination of both deoxynucleotide and ribonucleotide bases.

9. The method of claim 7, wherein said oligonucleotide comprises modified nucleotide bases.

10. The method of claim 7, wherein said oligonucleotide further comprises a moiety that is a member selected from:

biotin, a magnetic bead, a second oligonucleotide, an organic molecule, a polypeptide, a nucleic acid binding dye, a particle, an antibody.

11. The method of claim 1, wherein said treating step (e) comprises heating said amplified template nucleic acids to separate said circular template nucleic acids from said linear amplification products.

12. The method of claim 1, wherein said treating step (e) comprises applying an exonuclease such that said linear nascent strands are digested, leaving only said circular template nucleic acids.

13. The method of claim 1, wherein said amplifying step (b) is allowed to proceed for a sufficient amount of time such that said linear nascent strand comprises multiple copies of said sequences complementary to said one or more target sequences.