Abstract: The present invention provides novel engineered polypeptides that support both reverse transcription and DNA amplification in manganese-independent reactions. The present invention also provides methods for amplifying template nucleic acids using such polypeptides. This invention addresses deficiencies in the current state of the art in nucleic acid amplification-based detection of template nucleic acids, especially RNA targets, including deficiencies in detection sensitivity, specificity, enzyme stability, inhibitor tolerance and time to result compared with manganese-dependent thermostable reverse transcriptases and two-enzyme solutions.

Abstract: The present invention relates to a DNA and/or RNA ligase enzyme, its amino acid sequence, its nucleic acid sequence and to DNA and/or RNA ligase proteins encoded by these nucleic acid sequences, as well as nucleic acid or amino acid constructs comprising portions of the nucleic acid or amino acid sequence of the DNA and/or RNA ligase enzyme. The invention further relates to methods of using the DNA and/or RNA ligase enzyme for molecular biological assays and molecular diagnostic applications as well as to a kit containing the DNA and/or RNA ligase enzyme.

Abstract: The present invention relates to a polymerase enzyme with improved ability to incorporate reversibly terminating nucleotides. The enzyme comprising the following mutations in the motif A region (SGS). It relates to a polymerase enzyme according to SEQ ID NO. 1 with mutations in the amino acid sequence positions 409, 410 and 411.

Abstract: The present invention relates to a polymerase enzyme from Pyrococcus furiosus with improved ability to incorporate reversibly terminating nucleotides. The enzyme comprising the following mutations in the motif A region (SGS). It relates to a polymerase enzyme according to SEQ ID NO. 1 or any polymerase that shares at least 70% amino acid sequence identity thereto, comprising a mutation selected from the group of (i) at position 409 of SEQ ID NO. 3: serine (S) (L409S) and/or, (ii) at position 410 of SEQ ID NO. 3: glycine (G) (Y410G) and/or (iii) at position 411 of SEQ ID NO. 3: serine (S) (P411S), wherein the enzyme has little or no 3?-5? exonuclease activity.

Abstract: 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.

Abstract: The present invention provides novel engineered polypeptides that support both reverse transcription and DNA amplification in manganese-independent reactions. The present invention also provides methods for amplifying template nucleic acids using such polypeptides. This invention addresses deficiencies in the current state of the art in nucleic acid amplification-based detection of template nucleic acids, especially RNA targets, including deficiencies in detection sensitivity, specificity, enzyme stability, inhibitor tolerance and time to result compared with manganese-dependent thermostable reverse transcriptases and two-enzyme solutions.

Abstract: 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.

Abstract: 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.

Abstract: 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? 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 (Tm) of the primer at least 10° C. below the reaction temperature. The amplification is effected under isothermal condition.

Abstract: 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? 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 (Tm) of the primer at least 10° C. below the reaction temperature. The amplification is effected under isothermal condition.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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? 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 (Tm) of the primer at least 10° C. below the reaction temperature. The amplification is effected under isothermal condition.

Abstract: 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.

Abstract: 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? 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 (Tm) of the primer at least 10° C. below the reaction temperature. The amplification is effected under isothermal condition.

Abstract: 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? 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 (Tm) of the primer at least 10° C. below the reaction temperature. The amplification is effected under isothermal condition.