Methods, kits and solutions for preparing sample material for nucleic acid amplification
First Claim
1. A method for processing sample material for amplification of one or more selected target nucleic acid sequences contained therein, said method comprising the steps of:
- obtaining a sample of material potentially containing the target nucleic acid sequences;
mixing said sample with an extraction buffer solution, said extraction buffer solution comprising at least one detergent composition and at least one salt composition, said salt composition being present in a greater than 1 molar concentration;
centrifuging the mixture to obtain a first supernatant portion;
heating said first supernatant portion;
centrifuging said first supernatant portion to precipitate proteins and to obtain a second supernatant portion;
precipitating nucleic acids within said second supernatant portion; and
dissolving said nucleic acids.
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Accused Products
Abstract
The present invention provides methods and apparatus for detecting and discriminating multiple analytes within a test sample which are simple, user-friendly, cost-effective and fast. In particular, it is preferred that the overall time for sample preparation, nucleic acid sequence amplification, and nucleic acid sequence differentiation be about 5 hours or less. The methods of the present invention comprise (i) rapid sample processing means for rapidly preparing sample material of various types for amplification of nucleic acid sequences using unique nucleic acid extraction buffer formulations, (ii) multianalyte non-preferential amplifying process means for simultaneously and non-preferentially amplifying multiple target nucleic acid sequences, if present within the sample, using appropriate primer oligonucleotides optimized to achieve substantially similar amplification efficiencies, and (iii) multianalyte recognition process means for detecting and discriminating amplified nucleic acid sequences which incorporate nucleic acid sequence mismatch detection means for differentiating minor mismatches between multiple amplified nucleic acid sequences, including only single base mismatches, using appropriate probe oligonucleotides modified with neutral base substitution molecules. The processing kit products in accord with the present invention may incorporate all, or only some, of the above-described means.
117 Citations
30 Claims
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1. A method for processing sample material for amplification of one or more selected target nucleic acid sequences contained therein, said method comprising the steps of:
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obtaining a sample of material potentially containing the target nucleic acid sequences; mixing said sample with an extraction buffer solution, said extraction buffer solution comprising at least one detergent composition and at least one salt composition, said salt composition being present in a greater than 1 molar concentration; centrifuging the mixture to obtain a first supernatant portion; heating said first supernatant portion; centrifuging said first supernatant portion to precipitate proteins and to obtain a second supernatant portion; precipitating nucleic acids within said second supernatant portion; and dissolving said nucleic acids. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 22)
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- 20. A kit useful in the processing of sample material for amplification of one or more selected target nucleic acid sequences contained therein, said kit comprising an extraction buffer solution, said extraction buffer solution comprising two detergent compositions and at least one salt composition, said salt composition being present in a greater than 1 molar concentration.
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26. A solution useful in the processing of sample material for amplification of one or more selected target nucleic acid sequences contained therein, said solution comprising two detergent compositions and at least one salt composition, said salt composition being present in a greater than 1 molar concentration.
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27. A solution useful in the processing of sample material for amplification of one or more selected target nucleic acid sequences contained therein, said solution comprising 1% p-tert-octylphenoxypolyethoxyethanol detergent, 0.5% polyoxyethylenesorbitan monolaurate detergent, and 2.0 M NaCl.
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28. A solution useful in the processing of sample material for amplification of one or more selected target nucleic acid sequences contained therein, said solution comprising 1% t-octylphenoxypolyethoxyethanol detergent, 0.5% polyoxyethylenesorbitan monolaurate detergent, and 6.0 M NaCl.
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29. A solution useful in the processing of sample material for amplification of one or more selected target nucleic acid sequences contained therein, said solution comprising 1% t-octylphenoxypolyethoxyethanol detergent, 0.5% polyoxyethylenesorbitan monolaurate detergent, and 2.0 M NaCl.
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30. A solution useful in the processing of sample material for amplification of one or more selected target nucleic acid sequences contained therein, said solution comprising 1% p-tert-octylphenoxypolyethoxyethanol detergent, 0.5% polyoxyethylenesorbitan monolaurate detergent, and 3.0 M NaCl.
Specification