DIGITAL SEQUENCING USING MASS LABELS

• US 20180312916A1
• Filed: 04/14/2018
• Published: 11/01/2018
• Est. Priority Date: 04/26/2017
• Status: Active Application

• Current Assignee: Mike Joseph Pugia, Zane Baird, Zehui Cao
• Original Assignee: Mike Joseph Pugia, Zane Baird, Zehui Cao
• Inventors: Pugia, Mike Joseph, Baird, Zane, Cao, Zehui
• Application Number:
  US15/953,414
• Publication Number:
  US 20180312916A1
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

The invention provides a means to sequence a gene by mass spectroscopy by release and detection of mass labeled nucleic acids. Mass labels are designed as chain terminators nucleic acid and optimal for ionization by the mass spectrometric method used and there is no loss of sensitivity across genes sequenced and the amplification can be minimized.

Claims

1. A method of gene sequencing by mass spectroscopy said method comprising release and detection of mass labeled nucleic acids.

2. The method of claim 1, wherein the mass labels are attached as chain terminators to the nucleic acid.

3. The method of claim 1, wherein the mass labels include a breakable bond.

4. The method of claim 1, wherein the mass labels are optimized for ionization and detection by mass spectrometry.

5. The method of claim 1, wherein said mass labels are attached to the nucleic acids by conventional organic synthesis.

6. The method of claim 5, wherein said synthesis includes amplification of the nucleic acids and the releasable mass label terminators are 2′

,3′

dideoxynucleotides (ddNTPs).

7. The method of claim 5, wherein said synthesis of nucleic acids include lengths of <

3000 base pairs.

8. The method of claim 1, wherein the mass labels are attached to nucleic acids that are isolated.

9. The method of claim 8, wherein said nucleic acids are isolated by capture and purification.

10. The method of claim 8, wherein said nucleic acids are captured on particles or contained inside droplets.

11. The method of claim 8, wherein said captured nucleic acids are inside cells or released from cells.

12. The method of claim 10, wherein said captured nucleic acids are isolated by size exclusion filtration, or captured on particles.

13. The method of claim 1, wherein said detection further requires release from a liquid holding area for mass spectroscopic analysis.

14. The method of claim 1, wherein said measurement of nucleic acids by mass label can serve as a bar code to identify the presence of unique analyte or as a signal to quantitate the amount of analyte.

15. The method of claim 1, wherein said detection requires determining the number of base pairs in nucleic acids by mass and release of the mass label-terminator to identify the terminal nucleotide in the nucleic acids.

16. The method of claim 1, wherein said method further includes the following steps:

(a) isolation of the nucleic acid;

(b) amplification of the nucleic acid and chain termination with a 2′

,3′

dideoxynucleotides as the releasable mass label terminator;

(c) identification of the number of base pairs in the products by mass and;

(d) release and identification of the mass label-terminator to identify the terminal nucleotide in the sequence.