Abstract: Mutant polymerases are provided that have improved ability to incorporate modified nucleotides, including 3?-OH unblocked reversible terminators. The mutant polymerases may be used in a variety of applications, such as for polynucleotide sequencing, primer extension reactions, and template-independent enzymatic oligonucleotide synthesis.

Abstract: Mutant polymerases are provided that have improved ability to incorporate modified nucleotides, including 3?-OH unblocked reversible terminators. The mutant polymerases may be used in a variety of applications, such as for polynucleotide sequencing, primer extension reactions, and template-independent enzymatic oligonucleotide synthesis.

Abstract: Methods and compositions are provided for amplifying a pool of oligonucleotides, such as dual guide oligonucleotide constructs comprising sequences encoding a first guide RNA segment and a sequence encoding a second guide RNA segment. An amplification mixture is formed comprising the pool of oligonucleotides, an amplification enzyme, deoxyribonucleotide triphosphates, and primers. The amplification mixture is thermocycled a sufficient number of times and under conditions to produce a library of oligonucleotide constructs. The present methods and compositions provide dual guide libraries, including libraries that are essentially free of scrambled library members.

Abstract: Methods and compositions are described for multi-stage primer extension reactions such as multiplex polymerase chain reactions (PCR) and reverse transcriptase PCR. Primer extension stages are performed in a closed vessel without opening the vessel between stages. The multi-stage primer extension methods and compositions utilize earlier stage primers in an earlier stage and later stage primers in a later stage, wherein the later stage primers are blocked from extension during the earlier stage. The blocked primers of the present technology comprise photocleavable blocking groups and are substantially inactive until the blocking group is cleaved by exposure to ultraviolet light. The blocked primers can be activated by ultraviolet light without opening the vessel.

Abstract: Methods and compositions are provided for amplifying a pool of oligonucleotides, such as dual guide oligonucleotide constructs comprising sequences encoding a first guide RNA segment and a sequence encoding a second guide RNA segment. An amplification mixture is formed comprising the pool of oligonucleotides, an amplification enzyme, deoxyribonucleotide triphosphates, and primers. The amplification mixture is thermocycled a sufficient number of times and under conditions to produce a library of oligonucleotide constructs. The present methods and compositions provide dual guide libraries, including libraries that are essentially free of scrambled library members.

Abstract: This disclosure provides a method comprising: a) clamping the top and bottom strands of a double stranded DNA molecule to produce a duplex in which the top and bottom strands are linked; b) denaturing the duplex to produce a denatured product; and c) renaturing the denatured product in the presence of a labeled oligonucleotide that is complementary to a sequence of nucleotides in the double stranded DNA molecule, thereby producing a D-loop-containing product. Kits for performing the method and products made by the method are also provided.

Abstract: Provided herein, among other things, are a variety of methods for transposase-mediated tagging and amplification of short DNA fragments, e.g., between about 150 bp and 1.5 Kb in length. In some aspects, the method includes tagging the DNA fragments with a first primer sequence using barcoded transposases followed by a primer extension reaction to introduce a second primer sequence, e.g., using random or gene-specific primers. Kits for performing this method are also provided.

Abstract: Provided herein, among other things, are a variety of methods for transposase-mediated tagging and amplification of short DNA fragments, e.g., between about 150 bp and 1.5 Kb in length. In some aspects, the method includes tagging the DNA fragments with a first primer sequence using barcoded transposases followed by a primer extension reaction to introduce a second primer sequence, e.g., using random or gene-specific primers. Kits for performing this method are also provided.

Abstract: The invention relates to the generation and characterization of stable MMLV reverse transcriptase mutants. The invention also discloses methods of using stable MMLV reverse transcriptase mutants.

Abstract: The present invention provides mutants of DNA polymerases having an increased rate of incorporation of nucleotides into nucleic acids undergoing polymerization and having an enhanced resistance to inhibitors of DNA polymerase activity. The mutant polymerases are well suited for fast PCR applications, for PCR amplification of targets in samples that contain inhibitors of wild-type polymerases, and for fast PCR amplification of samples containing DNA polymerase inhibitors. In exemplary embodiments, the mutants are mutants of Taq DNA polymerase.

Abstract: The subject invention relates to compositions comprising an enzyme mixture which comprises a first enzyme and a second enzyme, where the first enzyme comprises a DNA polymerization activity and the second enzyme comprises an 5?-3? exonuclease activity and a reduced DNA polymerization activity. The invention also relates to the above compositions in kit format and methods for high fidelity DNA synthesis using the subject compositions of the invention.

Abstract: The invention relates to compositions and methods utilizing split polymerase enzymes composed of at least two discrete polypeptides that stably associate to form a single polymerase. The invention further relates to nucleic acid constructs for expressing the split polymerases of the invention, and methods for using the split polymerases of the invention. The enzymes of the invention are useful in many applications calling for the detectable labeling of nucleic acids and are particularly useful in quantitative PCR (QPCR) and DNA sequencing applications.

Abstract: The invention features a novel isolated Family B DNA polymerase, a Thermococcus polymerase JDF-3, and mutant recombinant forms thereof. Mutant polymerases of the invention are deficient in 3? to 5? exonuclease activity and/or exhibit reduced discrimination against non-conventional nucleotides relative to the wild-type form of the polymerase.

Abstract: This disclosure provides a method comprising: a) clamping the top and bottom strands of a double stranded DNA molecule to produce a duplex in which the top and bottom strands are linked; b) denaturing the duplex to produce a denatured product; and c) renaturing the denatured product in the presence of a labeled oligonucleotide that is complementary to a sequence of nucleotides in the double stranded DNA molecule, thereby producing a D-loop-containing product. Kits for performing the method and products made by the method are also provided.

Abstract: The invention features a novel isolated Family B DNA polymerase, a Thermococcus polymerase JDF-3, and mutant recombinant forms thereof. Mutant polymerases of the invention are deficient in 3? to 5? exonuclease activity and/or exhibit reduced discrimination against non-conventional nucleotides relative to the wild-type form of the polymerase.

Abstract: The invention provides novel extracts, proteins, and complexes that improve the polymerization activity of nucleic acid polymerases. Included within the aspects of the invention are methods for identifying compositions with a polymerase enhancing activity, methods for purifying and using these compositions, and specific extracts, proteins, and complexes that function to enhance polymerase activity. As an example, specifically described is nucleotide and amino acid sequence information for a Pyrococcus furiosus PEF (P45), which was used to produce a recombinant PEF.

Abstract: The invention provides novel extracts, proteins, and complexes that improve the polymerization activity of nucleic acid polymerases. Included within the aspects of the invention are methods for identifying compositions with a polymerase enhancing activity, methods for purifying and using these compositions, and specific extracts, proteins, and complexes that function to enhance polymerase activity. As an example, specifically described is nucleotide and amino acid sequence information for a Pyrococcus furiousus PEF (P45), which was used to produce a recombinant PEF.

Abstract: The subject invention relates to compositions comprising an enzyme mixture which comprises a first enzyme and a second enzyme, where the first enzyme comprises a DNA polymerization activity and the second enzyme comprises an 3?-5? exonuclease activity and a reduced DNA polymerization activity. The invention also relates to the above compositions in kit format and methods for high fidelity DNA synthesis using the subject compositions of the invention.

Abstract: The present invention provides mutants of DNA polymerases having an increased rate of incorporation of nucleotides into nucleic acids undergoing polymerization and having an enhanced resistance to inhibitors of DNA polymerase activity. The mutant polymerases are well suited for fast PCR applications, for PCR amplification of targets in samples that contain inhibitors of wild-type polymerases, and for fast PCR amplification of samples containing DNA polymerase inhibitors. In exemplary embodiments, the mutants are mutants of Taq DNA polymerase.

Abstract: Provided herein, among other things, are a variety of methods for transposase-mediated tagging and amplification of short DNA fragments, e.g., between about 150 bp and 1.5 Kb in length. In some aspects, the method includes tagging the DNA fragments with a first primer sequence using barcoded transposases followed by a primer extension reaction to introduce a second primer sequence, e.g., using random or gene-specific primers. Kits for performing this method are also provided.