MULTI-PRIMER AMPLIFICATION METHOD FOR BARCODING OF TARGET NUCLEIC ACIDS
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Abstract
In certain embodiments, the present invention provides amplification methods in which nucleotide tag(s) and, optionally, a barcode nucleotide sequence are added to target nucleotide sequences. In other embodiments, the present invention provides a microfluidic device that includes a plurality of first input lines and a plurality of second input lines. The microfluidic device also includes a plurality of sets of first chambers and a plurality of sets of second chambers. Each set of first chambers is in fluid communication with one of the plurality of first input lines. Each set of second chambers is in fluid communication with one of the plurality of second input lines. The microfluidic device further includes a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.
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Citations
83 Claims
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1-16. -16. (canceled)
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17. A method for amplifying a plurality of target nucleic acids in a plurality of samples, the method comprising:
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preparing an amplification mixture for each target nucleic acid, said amplification mixture comprising; a forward primer comprising a target-specific sequence; and a reverse primer comprising a target-specific sequence; subjecting each amplification mixture to amplification to produce a plurality of target nucleotide sequences; tagging the target nucleotide sequences to produce a plurality of target amplicons, each comprising first and/or second nucleotide tags flanking the target nucleotide sequence; wherein at least 50 percent of the target amplicons are present at greater than 50 percent of the average number of copies of target amplicons and less than 2-fold the average number of copies of target amplicons. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 26, 27, 30, 31, 38, 39, 53, 54, 55, 56)
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25. (canceled)
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28-29. -29. (canceled)
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32-37. -37. (canceled)
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40-52. -52. (canceled)
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57. A microfluidic device comprising:
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a plurality of first input lines; a plurality of second input lines; a plurality of sets of first chambers, wherein each set of first chambers is in fluid communication with one of the plurality of first input lines; a plurality of sets of second chambers, wherein each set of second chambers is in fluid communication with one of the plurality of second input lines; a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines; and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.
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58-65. -65. (canceled)
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66. A method of operating a microfluidic device having an assay chamber, a sample chamber, and a harvesting port, the method comprising:
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closing a fluid line between the assay chamber and the sample chamber; flowing a sample into the sample chamber via a sample input line; flowing an assay into the assay chamber via an assay input line; opening the fluid line between the assay chamber and the sample chamber; combining at least a portion of the sample and at least a portion of the assay to form a mixture; reacting the mixture to form a reaction product; closing the fluid line between the assay chamber and the sample chamber; flowing a harvesting reagent from the harvesting port to the sample chamber; and removing the reaction product from the microfluidic device.
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67-81. -81. (canceled)
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82. A method of preparing reaction products, the method comprising:
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providing M samples; providing N assays; mixing the M samples and N assays to form M×
N pairwise combinations, each of the M×
N pairwise combinations being contained in a closed volume;forming M×
N reaction products from the M×
N pairwise combinations; andrecovering the M×
N reaction products.
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83-95. -95. (canceled)
Specification