US5498392A — Mesoscale polynucleotide amplification device and…

Claim 1

Independent Claim 138 words

A device for amplifying a preselected polynucleotide in a sample by conducting a polynucleotide amplification reaction, the device comprising: a solid substrate which is microfabricated to define: a sample inlet port; a mesoscale flow system comprising: a sample flow channel extending from said inlet port; and a polynucleotide amplification reaction chamber, in fluid communication with said flow channel; said sample flow channel and said reaction chamber having at least one cross-sectional dimension of width or depth which is about 0.1 to 500 μm, said chamber and said channel being of dissimilar dimension; and a fluid exit port in fluid communication with said flow system; and means for thermally cycling the contents of said chamber whereby in each cycle the temperature is controlled to dehybridize double stranded polynucleotide and to permit the in vitro amplification of a preselected polynucleotide.

Claim 2

Independent Claim 138 words

A device for amplifying a preselected polynucleotide in a sample by conducting a polynucleotide amplification reaction, the device comprising: a solid substrate which is microfabricated to define: a sample inlet port; a mesoscale flow system comprising: a sample flow channel extending from said inlet port; and a polynucleotide amplification reaction chamber, in fluid communication with said flow channel; said sample flow channel and said reaction chamber having at least one cross-sectional dimension of width or depth which is about 0.1 to 500 μm, said chamber and said channel being of dissimilar dimension; and a fluid exit port in fluid communication with said flow system; and means for thermally cycling the contents of said chamber whereby in each cycle the temperature is controlled to dehybridize double stranded polynucleotide and to permit the in vitro amplification of a preselected polynucleotide.

Claim 3

Dependent Claim 129 words

The device of claim 1, wherein said amplification reaction is a polymerase chain reaction (PCR), and wherein said amplification chamber contains: a preselected polynucleotide, a polymerase, nucleoside triphosphates, a first primer hybridizable with said sample polynucleotide, and a second primer hybridizable with a nucleic acid comprising a sequence complementary to said polynucleotide, wherein said first and second primers define the termini of the polynucleotide product of the polymerization reaction; and wherein said means for thermally cycling comprises means for thermally cycling the contents of said chamber between a temperature controlled to dehybridize double stranded polynucleotide thereby to produce single stranded polynucleotide, to permit annealing of said primers to complementary regions of single stranded polynucleotide, and to permit synthesis of polynucleotide between said primers, thereby to amplify said preselected polynucleotide.

Claim 4

Dependent Claim 84 words

The device of claim 1 wherein said amplification chamber comprises: a first section at a temperature which dehybridizes double stranded polynucleotide; a second section at a temperature which permits annealing of complementary regions of single stranded polynucleotide and permits amplification of polynucleotide; a flow path disposed between said first and second sections; and wherein said device includes: means for repeatedly transporting the contents of said chamber between at least said first and said second sections to implement plural cycles of amplification of said polynucleotide.

Claim 5

Dependent Claim 61 words

The device of claim 3 wherein said second section and said flow path are spaced apart from said first section such that upon transport of the contents of said chamber from said first section to said second section, the sample cools passively to a temperature sufficient to permit annealing of single stranded polynucleotide and to permit amplification of said preselected polynucleotide.

Claim 6

Dependent Claim 18 words

The device of claim 3 further comprising means for separately thermally controlling said first and said second sections.

Claim 7

Dependent Claim 14 words

The device of claim 4 further comprising means for thermally controlling said first section.

Claim 8

Dependent Claim 20 words

The device of claim 5 or 6 wherein said means for thermally controlling comprises electrical resistance means in said substrate.

Claim 9

Dependent Claim 23 words

The device of claim 5 or 6 wherein said means for thermally controlling comprises means for providing electromagnetic energy to said amplification chamber.

Claim 10

Dependent Claim 12 words

The device of claim 1 wherein said solid substrate comprises microfabricated silicon.

Claim 11

Dependent Claim 36 words

The device of claim 1 further comprising an appliance for use in combination with said substrate, said appliance comprising: means for holding said substrate; and fluid input means interfitting with an inlet port on said substrate.

Claim 12

Dependent Claim 27 words

The device of claim 10 further comprising pump means for passing fluid through the flow system of said substrate when it is held in said holding means.

Claim 13

Dependent Claim 23 words

The device of claim 11 wherein said appliance further comprises a reagent reservoir and means for delivering a reagent to said flow system.

Claim 14

Dependent Claim 27 words

The device of claim 10 wherein said means for thermally cycling comprises an electromagnetic energy source; and wherein said electromagnetic energy source is provided in said appliance.

Claim 15

Independent Claim 138 words

A device for amplifying a preselected polynucleotide in a sample, the device comprising: a solid substrate microfabricated to define: a sample inlet port; a mesoscale flow system comprising: a sample flow channel extending from said inlet port; and a polynucleotide amplification reaction chamber, in fluid communication with said flow channel, containing a preselected polynucleotide and polynucleotide amplification reagents; said sample flow channel and said reaction chamber having at least one cross-sectional dimension of width or depth which is about 0.1 to 500 μm, said chamber and said channel being of dissimilar dimension; and a fluid exit port in fluid communication with said flow system; and means for thermally cycling the contents of said chamber whereby, in each cycle, temperature is controlled to dehybridize double stranded polynucleotide, and to permit synthesis of polynucleotide, thereby to amplify said preselected polynucleotide.

Claim 16

Independent Claim 138 words

A device for amplifying a preselected polynucleotide in a sample, the device comprising: a solid substrate microfabricated to define: a sample inlet port; a mesoscale flow system comprising: a sample flow channel extending from said inlet port; and a polynucleotide amplification reaction chamber, in fluid communication with said flow channel, containing a preselected polynucleotide and polynucleotide amplification reagents; said sample flow channel and said reaction chamber having at least one cross-sectional dimension of width or depth which is about 0.1 to 500 μm, said chamber and said channel being of dissimilar dimension; and a fluid exit port in fluid communication with said flow system; and means for thermally cycling the contents of said chamber whereby, in each cycle, temperature is controlled to dehybridize double stranded polynucleotide, and to permit synthesis of polynucleotide, thereby to amplify said preselected polynucleotide.

Claim 17

Dependent Claim 21 words

The device of claim 14 wherein said flow system further comprises a detection chamber in fluid communication with said amplification chamber.

Claim 18

Dependent Claim 82 words

The device of claim 14 wherein said amplification chamber comprises: a first section at a temperature which dehybridizes double stranded polynucleotide; a second section at a temperature which permits annealing of single stranded polynucleotide, and which permits amplification of polynucleotide; and a flow path disposed between said first and second sections; and wherein the device further comprises means for repeatedly transporting the contents of said chamber between said first and said second sections to implement plural cycles of amplification of said polynucleotide.

Claim 19

Dependent Claim 39 words

The device of claim 14 further comprising an appliance for use in combination with said substrate, said appliance comprising: a nesting site for holding said substrate, which comprises fluid input means interfitting with an inlet port on said substrate.

Claim 20

Dependent Claim 34 words

The device of claim 17 wherein said device includes electrical contacts fabricated in the substrate; and wherein said nesting site further includes an electrical connection for interfitting with said electrical contact in said substrate.

Claim 21

Dependent Claim 30 words

The device of claim 17 wherein the appliance further comprises pump means for passing fluid through the flow system of said substrate when it is held in said holding means.

Claim 22

Dependent Claim 13 words

The device of claim 17 wherein the appliance further comprises a power supply.

Claim 23

Independent Claim 171 words

A method for amplifying a preselected polynucleotide in a sample by conducting a polynucleotide amplification reaction, the method comprising: (i) providing a device comprising: a solid substrate microfabricated to define: a sample inlet port; a mesoscale flow system comprising: a sample flow channel extending from said inlet port; and a polynucleotide amplification reaction chamber in fluid communication with said flow channel; said sample flow channel and said reaction chamber having at least one cross-sectional dimension of width or depth which is about 0.1 to 500 μm, said chamber and said channel being of dissimilar dimension; and a fluid exit port in fluid communication with said flow system; and means for thermally regulating the contents of said chamber at a temperature controlled to permit amplification of said preselected polynucleotide; (ii) delivering, through said inlet port and said mesoscale flow system to said reaction chamber, a sample polynucleotide and reagents required for an in vitro polynucleotide amplification reaction; and (iii) thermally controlling the contents of said chamber to permit amplification of said polynucleotide.

Claim 24

Independent Claim 171 words

A method for amplifying a preselected polynucleotide in a sample by conducting a polynucleotide amplification reaction, the method comprising: (i) providing a device comprising: a solid substrate microfabricated to define: a sample inlet port; a mesoscale flow system comprising: a sample flow channel extending from said inlet port; and a polynucleotide amplification reaction chamber in fluid communication with said flow channel; said sample flow channel and said reaction chamber having at least one cross-sectional dimension of width or depth which is about 0.1 to 500 μm, said chamber and said channel being of dissimilar dimension; and a fluid exit port in fluid communication with said flow system; and means for thermally regulating the contents of said chamber at a temperature controlled to permit amplification of said preselected polynucleotide; (ii) delivering, through said inlet port and said mesoscale flow system to said reaction chamber, a sample polynucleotide and reagents required for an in vitro polynucleotide amplification reaction; and (iii) thermally controlling the contents of said chamber to permit amplification of said polynucleotide.

Claim 25

Dependent Claim 147 words

The method of claim 31 wherein said amplification reaction is a polymerase chain reaction (PCR); wherein, in step (i), said means for thermally controlling comprises means for thermally cycling the contents of said chamber; wherein step (ii) includes the step of adding to said amplification chamber: a polymerase, nucleoside triphosphates, a first primer hybridizable with said sample polynucleotide, and a second primer hybridizable with a nucleic acid comprising a sequence complementary to said polynucleotide, and wherein said first and second primers define the termini of the polynucleotide product of the polymerization reaction; and wherein step (iii) includes the step of thermally cycling the contents of said chamber whereby, in each cycle, the temperature is controlled to dehybridize double stranded polynucleotide thereby to produce single stranded polynucleotide, to permit annealing of complementary regions of single stranded polynucleotide, and to permit synthesis and polymerization of polynucleotide between said primers.

Claim 26

Dependent Claim 103 words

The method of claim 22 wherein said amplification chamber comprises: a first section at a temperature which dehybridizes double stranded polynucleotide; a second section at a temperature which permits annealing of complementary regions of single stranded polynucleotide and permits amplification of polynucleotide; a flow path disposed between said first and second sections; wherein the device further includes: means for repeatedly transporting the contents of said chamber between said first and said second sections; and wherein step (iii) includes the step of repeatedly transporting the contents of said chamber between said first and said second sections to implement plural cycles of amplification of polynucleotide.

Claim 27

Dependent Claim 89 words

The method of claim 23 wherein said first section is controlled at a temperature to dehybridize double stranded polynucleotide; and wherein said second section and said flow path are spaced apart from said first section such that upon transport of the contents of said chamber from said first section to said second section, the sample cools substantially to a temperature to anneal and polymerize; and wherein step (iii) includes the step of repeatedly transporting the contents of said chamber between said first and second sections to polymerize said polynucleotide.

Claim 28

Independent Claim 112 words

A device for amplifying a preselected polynucleotide in a sample by conducting a polynucleotide amplification reaction, the device comprising: a solid substrate microfabricated to define: a sample inlet port; a mesoscale flow system comprising: a sample flow channel extending from said inlet port; and a polynucleotide amplification chamber, in fluid communication with said flow channel, said chamber containing reagents for amplifying a preselected polynucleotide in vitro; said sample flow channel and said reaction chamber having at least one cross-sectional dimension of width or depth which is about 0.1 to 500 μm, said chamber and said channel being of dissimilar dimension; and a fluid exit port in fluid communication with said flow system.

Claim 29

Independent Claim 112 words

A device for amplifying a preselected polynucleotide in a sample by conducting a polynucleotide amplification reaction, the device comprising: a solid substrate microfabricated to define: a sample inlet port; a mesoscale flow system comprising: a sample flow channel extending from said inlet port; and a polynucleotide amplification chamber, in fluid communication with said flow channel, said chamber containing reagents for amplifying a preselected polynucleotide in vitro; said sample flow channel and said reaction chamber having at least one cross-sectional dimension of width or depth which is about 0.1 to 500 μm, said chamber and said channel being of dissimilar dimension; and a fluid exit port in fluid communication with said flow system.

Claim 30

Dependent Claim 16 words

The device of claim 25 wherein said reagents comprise reagents for conducting a polymerase chain reaction.

Claim 31

Dependent Claim 16 words

The device of claim 25 wherein said reagents comprise reagents for conducting a ligase chain reaction.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1999-03-12	1999-09-11	2000-03-12	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2003-03-12	2003-09-11	2004-03-12	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2007-03-12	2007-09-11	2008-03-12	$7700.00	Window Closed / Lapsed

Mesoscale polynucleotide amplification device and method

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