Spatially Encoded Biological Assays
The present invention provides assays and assay systems for use in spatially encoded biological assays. The invention provides an assay system comprising an assay capable of high levels of multiplexing where reagents are provided to a biological sample in defined spatial patterns; instrumentation capable of controlled delivery of reagents according to the spatial patterns; and a decoding scheme providing a readout that is digital in nature.
1 . (canceled)
2 . 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.
3 . The method of claim 2 , wherein the ligating in step (b) comprises the use of a ligase.
4 . The method of claim 3 , wherein the ligase is T4 DNA ligase.
5 . The method of claim 2 , wherein the method further comprises the use of one or both of an RNase and a protease.
6 . The method of claim 2 , 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.
7 . The method of claim 2 , wherein the amplifying in step (c) comprises the use of a DNA polymerase.
8 . The method of claim 2 , wherein step (d) comprises sequencing all or a portion of the sequence of the amplification product or a complement thereof.
9 . The method of claim 8 , wherein the sequencing is high-throughput sequencing.
10 . The method of claim 9 , wherein the high-throughput sequencing is digital nucleic acid sequencing or sequencing-by-ligation.
11 . The method of claim 2 , wherein the biological sample is affixed to a support.
12 . The method of claim 11 , wherein the support is a slide or a culture dish.
13 . The method of claim 2 , wherein the biological sample is a tissue sample.
14 . The method of claim 13 , wherein the tissue sample is a tissue section.
15 . The method of claim 14 , wherein the tissue section is a fresh, frozen tissue section.
16 . The method of claim 14 , wherein the tissue section is a fixed tissue section.
17 . The method of claim 16 , wherein the fixed tissue section is a formalin-fixed, paraffin-embedded (FFPE) tissue section.
18 . The method of claim 2 , 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.
19 . The method of claim 18 , 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.
20 . The method of claim 18 , wherein step (d) comprises sequencing all or a portion of the sequence of the second amplification product or a complement thereof.
21 . The method of claim 20 , wherein the sequencing is high-throughput sequencing.