The United States of America, As Represented by the Secretary, Department of Health and Human Services

Applied Biosystems, LLC

The United States of America As Represented by the Department of Health

Cytonix Corporation

Other

Jonathan E Silver

James F Brown

Stephen M Feinstone

Olga V Kalinina

Suzanne U Emerson

Vijaya R Keerikatte

Czeslaw Wychowski

A method for amplifying a target nucleic acid present in a sample includes introducing, via an entrance opening of a planar fluidic assembly, a sample containing one or more target nucleic acids into a flow channel of the planar fluidic assembly, the flow channel extending from the entrance opening to an exit vent and having a substantially uniform cross-section. A plurality of nucleic acid primers that are complementary to a portion of the one or more target nucleic acids are disposed at locations within and along the flow channel. The method further includes subjecting the sample introduced into the flow channel to a primer-based amplification reaction using the nucleic acid primers, wherein the primer-based amplification reaction produces amplified product of the one or more target nucleic acids, and retaining the amplified product at one or more of the locations within the flow channel during the primer-based amplification reaction. The method also includes detecting localized accumulation of the retained amplified product, the localized accumulation occurring at the one or more locations.

Device and method for amplifying target nucleic acid

A method comprises loading a sample portion into a sample chamber which comprises means for minimizing diffusion of the sample portion, subjecting the sample portion to an amplification step, and determining whether the sample portion contains at least one molecule of a target nucleic acid. If the sample portion contains a single molecule of the target nucleic acid, the sample portion would attain a detectable concentration of the target nucleic acid after a single round of amplification. Also, a microfluidic device comprising a sample portion and a sample chamber comprising means for minimizing diffusion of the sample portion. Also, a microfluidic device comprising a sample chamber and an amplification targeting reagent positioned in the first sample chamber.

Method for detecting the presence of a target nucleic acid sequence in a sample

A device for amplifying target nucleic acid in a sample can include a planar fluidic assembly including a transparent substrate, a porous material layer on a surface of the transparent substrate, and a cover over the porous material layer and sealingly affixed to the substrate. The cover may be spaced from the porous material layer and a flow channel defined between the porous material layer and the cover. The flow channel may have a uniform cross-section from a first end to a second end. The assembly can further include an inlet in flow communication with the first end of the flow channel to introduce sample containing target nucleic acid into the flow channel, an outlet in flow communication with the second end of the flow channel, and a plurality of nucleic acid primers retained by the porous material layer at discrete regions along and within the flow channel, each of the plurality of nucleic acid primers being complementary to a portion of the target nucleic acid in the sample to enable a primer-based amplification reaction of the target nucleic acid. The porous material layer may be configured to retain, at the discrete regions and during a primer-based amplification reaction, sample introduced to the flow channel and amplified product of the amplification reaction.

Device for amplifying target nucleic acid

A method comprising subjecting one or more sample portion(s) to a single amplification step, thereby amplifying a single molecule in the sample portion to a detectable level, and, in some embodiments, then determining whether the sample portion contains at least one molecule of the target nucleic acid. In some embodiments, the sample portion is in a porous sample structure, or in a sample chamber which comprises means for minimizing diffusion of the sample portion, or in a sample chamber which is inside a microcapillary device, or in a sample retaining means.

Method and device for detecting the presence of a single target nucleic acid in a sample

A method comprising loading a sample into a microfluidic device which comprises plural sample chambers, subdividing the sample into a plurality of sample portions, such that respective sample portions are positioned in each of a plurality of the sample chambers, and subjecting the sample portions loaded into the respective sample chambers to at least a first amplification step. Each of the sample chambers has a respective volume such that if a sample portion positioned in the sample chamber comprises at least one molecule of a target nucleic acid, the target nucleic acid would attain a detectable concentration in the sample chamber after a single round of amplification.

Method of loading sample into a microfluidic device

Method for detecting the presence of a single target nucleic acid in a sample

A miniaturized assembly is provided whereby a fluid sample can be divided into a plurality of sample portions in retaining wells and the sample fluid can be displaced from open ends of the wells while simultaneously being sealed in the wells. A method of dividing a fluid sample using the assembly is also provided.

A microfluidic device comprising a first surface portion and a first sample retaining element, which have differing affinities to a fluid, and a method comprising supplying a sample to such a device. In some embodiments, the differing affinity is a result of plasma, ion embedding, surface charging, chemical, optical, electronic and/or electromagnetic treatment. Also, a microfluidic device comprising at least one microcapillary device having a sample retaining element, at least one surface of which exhibits hydrophobicity, hydrophilicity, electromagnetic force exertion and electrostatic force exertion. Also, a microfluidic device comprising a first element having a hydrophilic pattern comprising at least a first sample retaining element. Also, a method comprising supplying a sample to a channel between a first element and a second element, and inducing in the first element at least one hydrophilic pattern by electrets or by internal or external electrodes to provide a charged surface.

Methods of detecting amplified product

Methods of detecting and quantifying genomic nucliec acid molecule sequences are provided using the simultaneous amplification of a plurality of discrete nanoliter-sized samples. A miniaturized closed assembly is also provided for carrying out amplification of a nucleic acid molecule by polymerase chain reaction in multiple nanoliter-sized samples. Methods of filling miniaturized sample chambers are also provided as are methods for determining the number of template molecules in a sample by conducting replicate nucleic acid sequence amplification reactions on a set of terminally diluted samples and counting the number of positive amplification reactions. The methods can be used to detect a single starting nucleic acid target molecule.

Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly

Methods of detecting and quantifying genomic nucleic acid molecule sequences are provided using the simultaneous amplification of a plurality of discrete nanoliter-sized samples. A miniaturized closed assembly is also provided for carrying out amplification of a nucleic acid molecule by polymerase chain reaction in multiple nanoliter-sized samples. Methods of filling miniaturized sample chambers are also provided as are methods for determining the number of template molecules in a sample by conducting replicate nucleic acid sequence amplification reactions on a set of terminally diluted samples and counting the number of positive amplification reactions. The methods can be used to detect a single starting nucleic acid target molecule.

Method of sampling, amplifying and quantifying segment of nucleic acid,

A method is described whereby new cDNA or RNA sequences can be introduced into or substituted for cDNA in any chosen position without specific sequence requirements using the polymerase chain reaction (PCR). The method entails the use of primers which are complementary to the 3' and 5' ends of the desired sequence to be inserted as well as to the 3' and 5' ends of the chosen site of insertion in the acceptor molecule. The desired sequence is amplified by PCR such that single stranded fragments are produced. The single stranded fragment of the desired sequence is then annealed to a single stranded acceptor molecule at the site of insertion and extended to produce a double stranded molecule. The double stranded molecule is then separated into two strands which are identical except that one of the strands contains the desired sequence inserted at the chosen site. A second double stranded molecule is then generated.

Techniques for producing site-directed mutagenesis of cloned DNA

A modification of the PCR technique is described which allows fragments of RNA or DNA to be amplified without prior knowledge of their sequence. The technique can be used to amplify viral nucleic acids present in small amounts in clinical material allowing, for example, the diagnosis of a particular virus infection or the discovery of new viruses.

Method for amplifying unknown nucleic acid sequences

A modification of the polymerase chain reaction (PCR) technique is described. The method allows the amplification of regions of DNA flanking a single region of known sequence, in contrast to standard PCR which requires two regions of known sequence at opposite ends of the fragment to be amplified. Various advantages of the new method are described.

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Bethesda, MD, United States of America

Jonathan E Silver