Nucleic acid template preparation for real-time pcr
AssigneeSamsung Techwin Co., Ltd.
InventorsJun LI, Jason OPDYKE
Life of this patent
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Abstract
The invention teaches a novel reagent formulation for the efficient preparation of a nucleic acid template from cells for high throughput real-time PCR analysis. The reagent permits rapid cell lysis and template preparation without the need for template purification and isolation. The reagent therefore dramatically improves throughput of real-time PCR analysis while at the same time permitting the rapid and sensitive real-time Catacleave PCR detection of a single molecule of nucleic acid template in as little as about 35 cycles of PCR amplification.
Claims41
A method of preparing a nucleic acid template for real-time PCR amplification, comprising the steps of:
- lysing cells in a lysis reagent comprising:
- a buffer with a pH of about 6 to about 9;
- a zwitterionic detergent at a concentration of about 0.125% to about 2%,
- an azide at a concentration of about 0.3 to about 2.5 mg/ml, and
- a protease,
- incubating the resulting cell lysate at about 55° C. for about 15 minutes to produce a substantially protein-free cell lysate,
- inactivating said protease at about 95° C. for about 10 minutes,
- adding said protein-free cell lysate directly to a real-time PCR amplification reaction mixture comprising:
- a pair of amplification primers that can anneal to a target DNA in said cell lysate;
- a probe comprising a detectable label and DNA and RNA nucleic acid sequences that are substantially complimentary to a region of the target DNA;
- an amplifying polymerase activity,
- an amplification buffer, and
- an RNase H activity,
- wherein, after amplification of the target DNA between the first and second amplification primers, the RNA sequences within the probe can form a RNA:DNA heteroduplex with the complimentary DNA sequences in said target DNA.
A method of preparing a nucleic acid template for real-time PCR amplification, comprising the steps of:
- lysing cells in a lysis reagent comprising:
- a buffer with a pH of about 6 to about 9;
- a zwitterionic detergent at a concentration of about 0.125% to about 2%, and
- an azide at a concentration of about 0.3 to about 2.5 mg/ml,
- incubating the cell lysate at about 55° C. for about 15 minutes,
- adding said cell lysate directly to an amplification reaction mixture comprising:
- a pair of amplification primers that can anneal to a target DNA in said cell lysate;
- a probe comprising a detectable label and DNA and RNA nucleic acid sequences that are substantially complimentary to a region of the target DNA;
- an amplifying polymerase activity,
- an amplification buffer, and
- an RNase H activity,
- wherein, after amplification of the target DNA between the first and second amplification primers, the RNA sequences within the probe can form a RNA:DNA heteroduplex with the complimentary DNA sequences in said target DNA.
A lysis reagent having:
- a buffer with a pH of about 6 to about 9;
- a zwitterionic detergent at a concentration of about 0.125% to about 2%, and
- an azide at a concentration of about 0.3 to about 2.5 mg/ml,
- wherein addition of about a two fold dilution of said reagent directly to an amplification reaction mixture comprising:
- a pair of amplification primers that can anneal to a target DNA in said cell lysate;
- a probe comprising a detectable label and DNA and RNA nucleic acid sequences that are substantially complimentary to a region of the target DNA;
- an amplifying polymerase activity,
- an amplification buffer, and
- an RNase H activity,
- does not inhibit said amplifying polymerase and RNase H enzymatic activities.
A lysis reagent having:
- a buffer with a pH of about 6 to about 9;
- a zwitterionic detergent at a concentration of about 0.125% to about 2%, and
- an azide at a concentration of about 0.3 to about 2.5 mg/ml, and
- a protease,
- wherein, after inactivation of said protease, addition of about a two fold dilution of said reagent directly to an amplification reaction mixture comprising:
- a pair of amplification primers that can anneal to a target DNA in said cell lysate;
- a probe comprising a detectable label and DNA and RNA nucleic acid sequences that are substantially complimentary to a region of the target DNA;
- an amplifying polymerase activity,
- an amplification buffer, and
- an RNase H activity,
- does not inhibit said amplifying polymerase and RNase H enzymatic activities.
Dependencies are inferred from the claim text.