Method and Kit for enhanced differential display
First Claim
1. Method for detecting different stages of cell development or detecting differences in gene expression comprising the steps of:
- a) contacting mRNA from each of said cell populations in separate reaction vessels with a first oligonucleotide primer, wherein said first oligonucleotide primer has a hybridizing sequence sufficiently complementary to a region of said mRNA to hybridize therewith,b) extending said first oligonucleotide primer in an extension reaction using the mRNA as a template to give a first DNA primer extension product complementary to the mRNA,c) contacting said first DNA primer extension product with a second oligonucleotide primer, wherein said second oligonucleotide primer has a hybridizing sequence sufficiently complementary to said first DNA primer extension product to hybridize therewith,d) extending said second oligonucleotide primer in an extension reaction using the first DNA primer extension product as a template to give a second DNA primer extension product complementary to the first DNA primer extension product,e) amplifying said first and second DNA primer extension products in a polymerase chain reaction comprising cycles of primer annealing, extension and denaturation, at an annealing temperature of between 35° and
45°
for at least two and not more than four cycles, thenf) amplifying said first and second DNA primer extension products at an annealing temperature of between 55° and
70°
for at least 16 cycles, to provide amplified gene sequencesg) separating said amplified gens sequences by size and/or charge; and
h) comparing amplified gens sequences separated in step (g) to detect an amplified gene sequence from one of said cell populations that is present at a different level in the other of said cell populations;
wherein said first and second oligonucleotide primers comprise at least 21 nucleotides.
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Abstract
An improved method for detecting and isolating differentially expressed mRNAs which comprises using first oligonucleotide primers for reverse transcription of mRNAs and both the first oligonucleotide primers and second oligonucleotide primers for amplification of the resultant cDNAs. The improvement of this method comprises providing first and second oligonucleotide primers with a length of at least 21 oligonucleotides. The method further comprises using a two-step PCR amplification, wherein non-stringent conditions are used for the first 1 to 4 cycles, and stringent conditions are used for the next 16 to 22 cycles. This highly reproducible method will permit the preparation of comprehensive catalogs of gene expression for any given cell type.
30 Citations
14 Claims
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1. Method for detecting different stages of cell development or detecting differences in gene expression comprising the steps of:
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a) contacting mRNA from each of said cell populations in separate reaction vessels with a first oligonucleotide primer, wherein said first oligonucleotide primer has a hybridizing sequence sufficiently complementary to a region of said mRNA to hybridize therewith, b) extending said first oligonucleotide primer in an extension reaction using the mRNA as a template to give a first DNA primer extension product complementary to the mRNA, c) contacting said first DNA primer extension product with a second oligonucleotide primer, wherein said second oligonucleotide primer has a hybridizing sequence sufficiently complementary to said first DNA primer extension product to hybridize therewith, d) extending said second oligonucleotide primer in an extension reaction using the first DNA primer extension product as a template to give a second DNA primer extension product complementary to the first DNA primer extension product, e) amplifying said first and second DNA primer extension products in a polymerase chain reaction comprising cycles of primer annealing, extension and denaturation, at an annealing temperature of between 35° and
45°
for at least two and not more than four cycles, thenf) amplifying said first and second DNA primer extension products at an annealing temperature of between 55° and
70°
for at least 16 cycles, to provide amplified gene sequencesg) separating said amplified gens sequences by size and/or charge; and h) comparing amplified gens sequences separated in step (g) to detect an amplified gene sequence from one of said cell populations that is present at a different level in the other of said cell populations; wherein said first and second oligonucleotide primers comprise at least 21 nucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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Specification