Systems and methods for whole genome amplification

• US 10,208,339 B2
• Filed: 02/18/2016
• Issued: 02/19/2019
• Est. Priority Date: 02/19/2015
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Mir, Alain-Albert, Schaal, Thomas David, Dunne, Jude, Srinivasan, Maithreyan
• Primary Examiner(s): Kim, Young J
• Application Number:
  US15/047,259
• Publication Number:
  US 20160304935A1
• Time in Patent Office: 1,097 Days
• Field of Search: None
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

Provided herein are systems and methods for whole genome amplification and sequencing. In particular, provided herein are systems and methods for detection of nucleic acid variants (e.g., rare variants) in limited samples.

Claims

1. A method of whole gene amplification (WGA), the method comprising:

a) combining;

a sample of genomic DNA template;

a phi29 polymerase;

nucleic acid primers; and

dNTPs;

into a reaction mixture under conditions sufficient for genomic DNA template-directed extension of the nucleic acid primers; and

b) amplifying the extended nucleic acid primers in the reaction mixture with a thermostable DNA polymerase to generate amplified whole genome DNA,

wherein said combining and amplifying are performed in a single vessel.

2. The method of claim 1, wherein said sample of genomic DNA is a single cell sample.

3. The method of claim 1, wherein said sample of genomic DNA is a plurality of cells.

4. The method of claim 1, further comprising the step of detecting one or more nucleic acid variants in said amplified whole genome DNA.

5. The method of claim 1, wherein said method further comprises the step of performing a nucleic acid sequencing assay on said amplified whole genome DNA.

6. The method of claim 1, wherein said amplification is conducted in a volume of 35 to 500 nL.

7. The method of claim 1, wherein at least a subset of said primers are specific for a nucleic acid target of interest.

8. The method of claim 1, wherein at least a subset of said primers comprises a sample barcode, molecular barcode, label, or tag sequence.

9. The method of claim 1, wherein said vessel is a well of a microfluidic chip comprising a plurality of wells.

10. The method of claim 9, wherein each of said wells comprises a distinct nucleic acid primer.