Compositions and methods for accurately identifying mutations

• US 10,011,871 B2
• Filed: 02/15/2013
• Issued: 07/03/2018
• Est. Priority Date: 02/17/2012
• Status: Active Grant

• Current Assignee: Fred Hutchinson Cancer Center
• Original Assignee: Fred Hutchinson Cancer Research Center (Fred Hutchinson Cancer Research Center|Argus Genetics|Mars)
• Inventors: Bielas, Jason H.
• Primary Examiner(s): Weiler, Karen S.
• Application Number:
  US14/378,870
• Publication Number:
  US 20150024950A1
• Time in Patent Office: 1,964 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 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/119 Double strand sequencing
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present disclosure provides compositions and methods for accurately detecting mutations by uniquely tagging double stranded nucleic acid molecules with dual cyphers such that sequence data obtained from a sense strand can be linked to sequence data obtained from an anti-sense strand when sequenced, for example, by massively parallel sequencing methods.

Claims

1. A double-stranded nucleic acid molecule library, comprising a plurality of target nucleic acid molecules and a plurality of random double-stranded cyphers,

wherein the nucleic acid library comprises nucleic acid molecules having a formula of 5′

-RE1-PS1-X^a-PS2-Y-PS2-X^b-PS1-RE2-3′

,

wherein RE1 is a first restriction endonuclease recognition sequence, PS1 is a first nucleic acid molecule priming site, PS2 is a second nucleic acid molecule priming site, RE2 is a second restriction endonuclease recognition sequence, Y comprises a target nucleic acid molecule, and X^a and X^b each comprise a first and second random cypher, respectively, wherein each cypher has a length ranging from about 6 nucleotides to about 9 nucleotides or has a length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides, and

wherein the double-stranded X^a cypher is identical to the X^b cypher for one or more target nucleic acid molecules, provided that the double-stranded cypher for each target nucleic molecule is different.

2. The double-stranded nucleic acid library of claim 1 wherein the plurality of double-stranded random cyphers each have the same number of nucleotides and comprise a length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 150 nucleotides.

3. The double-stranded nucleic acid library of claim 1 wherein the target nucleic acid molecules comprise a length ranging from about 10 nucleotides to about 10,000nucleotides or a length ranging from about 100 nucleotides to about 1,000 nucleotides.

4. The double-stranded nucleic acid library of claim 1 wherein the first restriction endonuclease recognition sequence RE1 and the second restriction endonuclease recognition sequence RE2 are the same.

5. The double-stranded nucleic acid library of claim 1 wherein the nucleic acid molecules having a formula of 5′

-RE1-PS1-X^a-PS2-Y-PS2-X^b-PS1-RE2-3′

are each contained in a self-replicating vector.

6. The double-stranded nucleic acid library of claim 5 wherein the self-replicating vector is a plasmid, cosmid, YAC, or viral vector.