Granted Patent B2

US 12,747,467 · App. 18/100,127 · Granted Sep 29, 2026

Inventor: Mark S. Chee (San Diego, CA)

C12Q1/6837B01L3/502715C12Q1/68C12Q1/6804C12Q1/6809C12Q1/6834C12Q1/6841C12Q1/6869C12Q1/6874C12Q1/6876C40B30/04C40B60/04G01N33/5308G01N33/543G01N33/54366G01N33/6845G01N33/6848C12Q1/6858C12Q2600/156C12Q2600/158G01N2458/10G01N2458/40

Abstract

Claims (38)

1 . A method of detecting a mitochondrial DNA at a location in a biological sample comprising:

(a) contacting the biological sample comprising mitochondrial DNA with a first probe and a second probe, wherein:

the first probe comprises a first sequence that hybridizes to a first portion of the mitochondrial DNA and a first universal priming site;

the second probe comprises a second sequence that hybridizes to a second portion of the mitochondrial DNA and a second universal priming site;

the first probe and the second probe hybridized to the mitochondrial DNA are capable of being ligated together; and

one or both of: (i) the first probe further comprises a nucleic acid sequence identifying the location to where the first probe was delivered and (ii) the second probe further comprises a nucleic acid sequence identifying the location to where the second probe was delivered;

(b) ligating the first probe and the second probe together to generate a ligation product;

(c) amplifying the ligation product using a first amplification primer that is capable of hybridizing to the first universal priming site, and a second amplification primer that is capable of hybridizing to the second universal priming site, to produce an amplification product; and

(d) determining all or a portion of the sequence of the amplification product or a complement thereof, and using the determined sequence to detect the mitochondrial DNA at the location in the biological sample.

2 . The method of claim 1 , wherein the ligating in step (b) comprises the use of a ligase.

3 . The method of claim 2 , wherein the ligase is T4 DNA ligase.

4 . The method of claim 1 , wherein the method further comprises the use of one or both of an RNase and a protease.

5 . The method of claim 1 , wherein the method further comprises, between steps (a) and (c), washing away the first probe and the second probe not hybridized to the mitochondrial DNA.

6 . The method of claim 1 , wherein the amplifying in step (c) comprises the use of a DNA polymerase.

7 . The method of claim 1 , wherein step (d) comprises sequencing all or a portion of the sequence of the amplification product or a complement thereof.

8 . The method of claim 7 , wherein the sequencing is high-throughput sequencing.

9 . The method of claim 8 , wherein the high-throughput sequencing is digital nucleic acid sequencing or sequencing-by-ligation.

10 . The method of claim 1 , wherein the biological sample is affixed to a support.

11 . The method of claim 10 , wherein the support is a slide or a culture dish.

12 . The method of claim 1 , wherein the biological sample is a tissue sample.

13 . The method of claim 12 , wherein the tissue sample is a tissue section.

14 . The method of claim 13 , wherein the tissue section is a fresh, frozen tissue section.

15 . The method of claim 13 , wherein the tissue section is a fixed tissue section.

16 . The method of claim 15 , wherein the fixed tissue section is a formalin-fixed, paraffin-embedded (FFPE) tissue section.

17 . The method of claim 1 , wherein:

the biological sample further comprises a second mitochondrial DNA at a different location in the biological sample;

step (a) further comprises contacting the biological sample with a third probe and a fourth probe, wherein:

the third probe comprises a first sequence that hybridizes to a first portion of the second mitochondrial DNA and the first universal priming site;

the fourth probe comprises a second sequence that hybridizes to a second portion of the second mitochondrial DNA and the second universal priming site;

the third probe and the fourth probe hybridized to the second mitochondrial DNA are capable of being ligated together; and

one or both of: (i) the third probe further comprises a nucleic acid sequence identifying the location to where the third probe was delivered and (ii) the fourth probe further comprises a nucleic acid sequence identifying the location to where the fourth probe was delivered;

step (b) further comprises ligating the third probe and the fourth probe together to generate a second ligation product;

step (c) further comprises amplifying the second ligation product using the first amplification primer and the second amplification primer to produce a second amplification product; and

step (d) further comprises determining all or a portion of the sequence of the second amplification product or a complement thereof, and using the determined sequence to detect the second mitochondrial DNA at a location in the biological sample.

18 . The method of claim 17 , wherein the method further comprises, between steps

(a) and (c), washing away the third probe and the fourth probe not hybridized to the second mitochondrial DNA.

19 . The method of claim 17 , wherein step (d) comprises sequencing all or a portion of the sequence of the second amplification product or a complement thereof.

20 . The method of claim 19 , wherein the sequencing is high-throughput sequencing.

Assignments (1)

Continuity (12)

References Cited (400)