Systems and methods for rapid identification of nucleic acid variants

• US 8,551,738 B2
• Filed: 11/11/2009
• Issued: 10/08/2013
• Est. Priority Date: 07/21/2005
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Ecker, David J., Hofstadler, Steven A., Hall, Thomas A., Sannes-Lowery, Kristin
• Primary Examiner(s): WILDER, CYNTHIA B
• Application Number:
  US12/616,422
• Publication Number:
  US 20100070194A1
• Time in Patent Office: 1,427 Days
• Field of Search: 435/283.1, 435/6, 435/91.2, 435/6.1, 435/92.1
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/10 the purpose being quantitative analysis
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

There is a need for nucleic acid analysis which is both specific and rapid, and in which no nucleic acid sequencing is required. The present invention addresses this need, among others by providing a method of nucleic acid amplification of overlapping sub-segments of a nucleic acid followed by molecular mass measurement of resulting amplification products by mass spectrometry, and determination of the base compositions of the amplification products.

Claims

1. A system, comprising:

a) at least one mass spectrometer having a component that measures-molecular masses of overlapping amplification products representing sub-segments of a nucleic acid;

b) at least one computer comprising a processor;

c) at least one database of base compositions of overlapping amplification products on a computer readable medium wherein said base compositions identify the number but not the nucleic acid gene sequence order of A residues, C residues, T residues, G residues and analogs thereof in a plurality of said overlapping amplification products; and

d) software that directs said processor to calculate base compositions of said overlapping amplification products from said molecular masses wherein said base compositions identify the number but not the nucleic acid gene sequence order of A residues, C residues, T residues, G residues and analogs thereof in said amplification products, and that identifies a source of said nucleic acid from said base compositions by comparing said determined base compositions to base compositions in said at least one database.

2. The system of claim 1, wherein said mass spectrometer is an electrospray mass spectrometer.

3. The system of claim 1, comprising at least one liquid handling robot.

4. The system of claim 1, comprising at least one thermal cycler.

5. The system of claim 1, wherein said at least one database contains base compositions of reference nucleic acids and/or sub-segments thereof.

6. The system of claim 5, wherein said base compositions of said reference nucleic acids in said at least one database and/or said sub-segments thereof correspond to overlapping amplification products representing sub-segments of mitochondrial DNA.

7. A method of analyzing data using the system of claim 1, the method comprising:

receiving data comprising said molecular masses of said overlapping amplification products representing sub-segments of said nucleic acid at said computer;

determining base compositions of said overlapping amplification products representing sub-segments of said nucleic acid from said molecular masses; and

,

identifying a source of said nucleic acid from said base composition of said overlapping amplification products representing sub-segments of said nucleic acid, using said database.

8. The method of claim 7, wherein said amplification products are from about 40 nucleobases in length to about 150 nucleobases in length.

9. The method of claim 7, wherein said nucleic acid is selected from the group consisting of:

a human chromosomal nucleic acid, a human mitochondrial nucleic acid, a bacterial nucleic acid, a viral nucleic acid, a fungal nucleic acid, a synthetic nucleic acid, a recombinant nucleic acid and a combination thereof.

10. The method of claim 7, wherein said identifying step identifies at least one amplification product whose base composition differs from the base composition of a corresponding reference sub-segment, thereby identifying a genetically-engineered bacterium, virus or fungus.

11. The method of claim 7, wherein said nucleic acid comprises at least a portion of an HV1 segment and/or an HV2 segment of a mitochondrial DNA.

12. A base composition analysis method using the system of claim 1, the method comprising:

(a) generating at least two of said overlapping amplification products from at least two sub-segments of said nucleic acid;

(b) measuring said molecular masses of said overlapping amplification products from said at least two sub-segments of said nucleic acid using a mass spectrometer;

(c) converting said measured molecular masses of said overlapping amplification products from said at least two sub-segments of said nucleic acid to base compositions; and

, (d) comparing said base compositions of said overlapping amplification products from said at least two sub-segments of said nucleic acid with said database of base compositions to identify a source of the nucleic acid.

13. The method of claim 12, comprising performing one or more of (a)-(d) in silico.

14. The method of claim 12, wherein said amplification products are from about 40 nucleobases in length to about 150 nucleobases in length.

15. The method of claim 12, wherein said nucleic acid is selected from the group consisting of:

a human chromosomal nucleic acid, a human mitochondrial nucleic acid, a bacterial nucleic acid, a viral nucleic acid, a fungal nucleic acid, a synthetic nucleic acid, a recombinant nucleic acid and a combination thereof.

16. The method of claim 12, wherein said identifying step identifies at least one amplification product whose base composition differs from the base composition of a corresponding reference sub-segment, thereby identifying a genetically-engineered bacterium, virus or fungus.

17. The method of claim 12, wherein said nucleic acid comprises at least a portion of an HV1 segment and/or an HV2 segment of a mitochondrial DNA.