In situ transcription in cells and tissues
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsLaurence Tecott, Jack D. Barchas, James Eberwine
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Abstract
In situ transcription is provided by hybridizing cells or tissue with a primer, and extending the primer bound to any template mRNA or DNA with transverse transcriptase or DNA polymerase in the presence of labeled nucleotides, which allows for detection of cells containing the template. The resulting cDNA may be eluted and used in a polymerase chain reaction for isolation of the DNA, and/or the cDNA electrophoresed, which may provide for information concerning the sequence, or the like.
Claims8
A method for producing a DNA strand intracellularly in eukaryotic or prokaryotic cells or tissues in a tissue section, said method comprising:
- contacting said cells or tissue in said tissue section under hybridizing conditions with a primer for a nucleic acid sequence of interest;
- and
- contacting said cells or tissue containing said primer in said tissue section with a reverse transcriptase and deoxynucleotide triphosphates under conditions for primer extension to produce a DNA copy of RNA to which said primer is bound.
A method for producing cDNA from mRNA intracellularly, said method comprising:
- contacting tissue suspected of containing said mRNA with a primer complementary to a sequence of said mRNA proximal to the 3' region of said mRNA under hybridizing conditions;
- contacting said primer-containing tissue with reverse transcriptase and deoxynucleotide triphosphates under conditions for primer extension and formation of cDNA, wherein at least one of said primer or said triphosphates is labeled with a label capable of providing a detectable signal;
- eluting said cDNA from said cells and isolating said cDNA substantially free of other nucleic acid by means of said label;
- and
- amplifying said cDNA under polymerase chain reaction conditions.
Dependencies are inferred from the claim text.