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US 12,590,302 B2Granted
Multiplexed sequencing in cells and tissues
AssigneeSingular Genomics Systems, Inc.
InventorsEli N. Glezer, Niek Van Wietmarschen, Daan Witters
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Abstract
Disclosed herein, inter alia, are methods and compositions for detecting a plurality of nucleic acids in a sample including a cell or tissue. The methods may include amplifying nucleic acid molecules in the sample and detecting the amplified nucleic acid molecules in the sample.
Claims23
A method of detecting different nucleic acid molecules in a sample comprising a cell or tissue, said method comprising:
- amplifying a plurality of different nucleic acid molecules in said cell or tissue to generate a first, a second, a third, and a fourth amplification product in said cell or tissue;
- binding a first primer to a first amplification product in said cell or tissue and incorporating 5 to 200 labeled nucleotides into the first primer and detecting the labeled nucleotides incorporated into the first primer;
- followed by
- binding a second primer to a second amplification product in said cell or tissue and incorporating 5 to 200 labeled nucleotides into the second primer and detecting the labeled nucleotides incorporated into the second primer;
- followed by
- binding a third primer to a third amplification product in said cell or tissue and incorporating 5 to 200 labeled nucleotides into the third primer and detecting the labeled nucleotides incorporated into the third primer;
- followed by
- binding a fourth primer to a fourth amplification product in said cell or tissue and incorporating 5 to 200 labeled nucleotides into the fourth primer and detecting the labeled nucleotides incorporated into the fourth primer;
- wherein the first, second, third, and fourth primers each comprise a different sequence.
6 more elements
A method of detecting different nucleic acid molecules in a tissue sample, said method comprising:
- amplifying a plurality of different circular polynucleotides in said tissue sample to generate a plurality of amplification products;
- binding a first primer to a first amplification product and incorporating a first labeled nucleotide into the first primer and incorporating an irreversibly terminated nucleotide into the first primer;
- binding a second primer to a second amplification product and incorporating a second labeled nucleotide into the second primer and incorporating an irreversibly terminated nucleotide into the second primer;
- and
- detecting the first and second incorporated nucleotides.
2 more elements
A method of detecting different nucleic acid molecules in a sample comprising a cell or tissue, said method comprising:
- amplifying a plurality of different nucleic acid molecules in said sample to generate a plurality of amplification products in said sample;
- binding a first primer to a first amplification product and incorporating a first labeled nucleotide into the first primer and detecting the labeled nucleotide;
- binding a second primer to a second amplification product and incorporating a second labeled nucleotide into the second primer and detecting the second labeled nucleotide;
- binding a third primer to a third amplification product and incorporating a third labeled nucleotide into the third primer and detecting the third labeled nucleotide;
- and
- binding a fourth primer to a fourth amplification product and incorporating a fourth labeled nucleotide into the fourth primer and detecting the fourth labeled nucleotide;
- wherein the first, second, third, and fourth primer are each a different sequence selected from SEQ ID NO:1 to SEQ ID NO:96.
4 more elements
A method of selectively sequencing nucleic acids in a cell or tissue, the method comprising:
- generating a plurality of amplification products in the cell or tissue, wherein a first subset of the amplification products comprises a first primer binding sequence, a second subset of the amplification products comprises a second primer binding sequence, a third subset of the amplification products comprises a third primer binding sequence, and a fourth subset of the amplification products comprises a fourth primer binding sequence;
- hybridizing a sequencing primer complementary to the first sequencing primer binding sequence of the first subset and sequencing a first sequence of the amplification products;
- followed by
- hybridizing a sequencing primer complementary to the second sequencing primer binding sequence of the second subset and sequencing a second sequence of the amplification products;
- followed by
- hybridizing a sequencing primer complementary to the third sequencing primer binding sequence of the third subset and sequencing a third sequence of the amplification products;
- followed by
- hybridizing a sequencing primer complementary to the fourth sequencing primer binding sequence of the fourth subset and sequencing a fourth sequence of the amplification products;
- and
- removing each sequencing primer prior to hybridizing a subsequent sequencing primer.
7 more elements
Dependencies are inferred from the claim text.