Multiplexed single molecule RNA visualization with a two-probe proximity ligation system
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsNikolay Samusik, Felice Alessio Bava, Yury Goltsev, Garry P. Nolan
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Abstract
SNAIL provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, e.g. at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells/second. The methods require only two primers for amplification, and may further include a detection primer.
Claims16
A method for determining the level of a target nucleic acid in a single cell, the method comprising:
- i) contacting a fixed and permeabilized cell with a pair of SNAIL oligonucleotide primers under conditions permissive for specific hybridization of the pair of SNAIL oligonucleotide primers to the target nucleic acid, wherein the pair of SNAIL oligonucleotide primers comprises a Splint Primer Oligonucleotide (SPO) and a Padlock Oligonucleotide (PO), wherein each of SPO and PO comprises a first complementarity region complementary to adjacent sequences on the target nucleic acid, wherein the first complementarity region of SPO is CR1 and the first complementarity region of PO is CR1′;
- and each of SPO and PO further comprises a second complementarity region, wherein the second complementarity region of SPO is CR2 located adjacent to CR1 and the second complementarity region of PO is CR2′ located adjacent to CR1′, wherein CR2′ is a split region of PO such that after said contacting a fixed and permeabilized cell with the pair of SNAIL oligonucleotide primers, the 5′ and the 3′ ends of PO hybridize to CR2 and the 5′ and the 3′ ends of PO are positioned on CR2 directly adjacent to one another, wherein the fixed and permeabilized cell is a single cell;
- ii) washing the cell to remove the pair of SNAIL oligonucleotide primers unbound to the target nucleic acid,
- iii) after the washing step, contacting the cell with a ligase such that the 5′ and the 3′ ends of the PO hybridized to the target nucleic acid are ligated to each other and generating a closed circle;
- iv) performing a rolling circle amplification using the closed circle as a template and SPO as a primer and generating an amplification product;
- v) contacting the amplification product with a detection probe under conditions permissive for specific hybridization of the detection probe to the amplification product;
- and
- vi) determining the level of the target nucleic acid in the single cell by detecting the level of the detection probe bound to the amplification product.
Dependencies are inferred from the claim text.