General Electric Company

Ge Healthcare Bio-sciences Corp.

Global Life Sciences Solutions USA LLC

Global Life Sciences Solutions Operations UK Ltd

Ryan Charles Heller

John Richard Nelson

Erik Leeming Kvam

Gregory Andrew Grossmann

Robert Scott Duthie

Alison Myfanwy Wakefield

Paresh Lakhubhai Patel

Stephen James Capper

Christopher Michael Puleo

Erin Jean Finehout

Nichole Lea Wood

William Patrick Waters

Peter James Tatnell

Wei Gao

Michael James Rishel

Jeffrey Kenneth Horton

Sonali Jagdish Shah

Nam Tran

Klaus Gustav Hentrich

Qingyan Au

Nicholas Hoe

A method is provided for generating single-stranded DNA circles from a biological sample. The method comprises the steps of: treating the biological sample with an extractant to release nucleic acids, thereby forming a sample mixture; neutralizing the extractant; denaturing the released nucleic acids to generate single-stranded nucleic acids; and contacting the single-stranded nucleic acids with a ligase that is capable of template-independent, intramolecular ligation of single-stranded DNA to generate the single-stranded DNA circles. All the steps of the method are performed without any intermediate nucleic acid isolation or nucleic acid purification. The single-stranded DNA circles may be amplified and further analyzed. Also provided is a kit which comprises compositions for carrying out the novel methods.

Method for nucleic acid analysis directly from an unpurified biological sample

Provided herein are methods for the collection and amplification of circulating nucleic acids from a non-celluar fraction of a biological sample. Circulating nucleic acids are extracted from the non-cellular fraction and are circularized to generate single-stranded nucleic acid circles, which are then subsequently amplified by rolling circular amplification using random primers to produce an amplified library. Devices for the collection of a non-cellular fraction from a biological sample are also provided. The device includes a filtration membrane and a dry solid matrix, which is in direct contact with the filtration membrane.

Method and device for collection and amplification of circulating nucleic acids

Provided herein are methods for generation and amplification of a single-stranded DNA circle in a single reaction vessel from a linear DNA without any intervening purification steps. The single-stranded DNA circle is generated via a template-independent single-stranded DNA ligation. Whole-genome amplification of linear chromosomal DNA in a single tube using ligation-assisted DNA amplification is also provided.

Ligase-assisted nucleic acid circularization and amplification

Provided herein are methods and kits for isothermal nucleic acid amplifications that use a target nucleic acid template; a reaction mixture comprising a DNA polymerase having a strand displacement activity, a deoxyribonucleoside triphosphate (dNTP) mixture, a primer with a 3' end and a 5&#x2032; end, a molecular crowding reagent, and a buffer solution for amplifying the target nucleic acid template. The buffer solution maintains a low salt concentration of the reaction mixture, and wherein the salt concentration results in a melting temperature (T) of the primer at least 10&#xb0; C. below the reaction temperature. The amplification is effected under isothermal condition.

Isothermal amplification under low salt condition

The invention discloses a method for determining a nucleotide sequence of a nucleic acid segment present in a biological sample, comprising the steps of: a) generating a fluorescent image of the sample by a protocol comprising immunofluorescence detection of at least five different target proteins in the sample; b) selecting a region of interest of the sample by comparing the image to a predetermined criterion; c) removing a subsample from the region of interest, and; d) determining a nucleotide sequence of a nucleic acid segment present in the subsample.

Method of determining a nucleotide sequence of a nucleic acid segment from a region of interest in a biological sample

Solid phase isothermal amplification

Provided herein are methods and kits for isothermal nucleic acid amplifications that use an oligocation-oligonucleotide conjugate primer for amplifying a target nucleic acid to generate amplicons. Isothermal DNA amplification methods that employ a strand displacing DNA polymerase and polyamine-oligonucleotide conjugate primer are also provided.

Isothermal amplification using oligocation-conjugated primer sequences

A method of amplifying RNA template is provided. The method comprises reverse-transcribing a ribonucleic acid (RNA) template to form a cDNA using a first reaction mixture comprising RNA template, at least one primer capable of hybridizing to the RNA template, a reverse transcriptase and deoxynucleoside triphosphates (dNTPs); and amplifying the cDNA to form an amplified product using a second reaction mixture comprising at least one strand displacement DNA polymerase, at least one inosine-containing primer and a nuclease that is capable of nicking DNA 3' to an inosine residue of the primer. The method is accomplished under an isothermal condition without denaturing the cDNA template. A method of quantifying RNA template in a sample and a method of detecting RNA template in a sample are also provided.

Method for isothermal DNA amplification starting from an RNA template

Provided herein are methods for the collection and amplification of circulating nucleic acids from a non-cellular fraction of a biological sample. Circulating nucleic acids are extracted from the non-cellular fraction and are circularized to generate single-stranded nucleic acid circles, which are then subsequently amplified by rolling circular amplification using random primers to produce an amplified library. Devices for the collection of a non-cellular fraction from a biological sample are also provided. The device includes a filtration membrane and a dry solid matrix, which is in direct contact with the filtration membrane.

Methods and kits for efficient amplification of nucleic acids are provided. The disclosure generally relates to methods and kits for nucleic acid amplification of target nucleic acids of interest. The methods described herein promote the synthesis of the target nucleic acid (i.e., template nucleic acid) by reducing the production of undesirable primer-dimer structures and chimeric nucleic acid products during the amplification process by using novel modified primers.

Methods and kits for reducing non-specific nucleic acid amplification

Provided herein are methods for generation and amplification of a single-stranded DNA circle in a single reaction vessel from a linear DNA without any intervening purification steps. The single-stranded DNA circle is generated via a template-independent single-stranded DNA ligation. Whole-genome amplification of circulating nucleic acids extracted from blood is provided. Kits for performing the disclosed methods are also provided.

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Guilderland, NY, United States of America

Ryan Charles Heller