Abstract: Disclosed herein are improved methods for the de novo synthesis of nucleic acids by cyclic extension using a template-independent polymerase. Secondary structure formation in the nascent chain, which may inhibit extension reactions, is suppressed by the use of monomers with O-alkylated nucleobases that prevent Watson-Crick base pairing and/or other structures. After the synthesis is completed, the nucleobases can be converted back into native form by enzymatic removal of the alkyl groups.

Abstract: Methods are disclosed for high throughput identification of optimized template-independent polymerase variants. The methods comprise generation of polymerase gene-encoding variants, isolation of variants, expression of variants, exposing expressed polymerase variants to desired conditions for activity, and identification of active variants, such as by hybridization to synthesized polynucleotides or by amplification. Encoding genes of active variants can be sequenced. The method can be performed iteratively to enhance generation and selection of preferred variants.

Abstract: The present disclosure provides modified terminal deoxynucleotidyl (TdT) polymerases and compositions (e.g., conjugates) comprising modified TdT polymerases. The present disclosure also provides circularly permuted terminal deoxynucleotidyl (TdT) polymerases as well as modified TdT polymerases and compositions (e.g., conjugates) comprising such cpTdT or modified TdT polymerases. Also provided are nucleic acids, vectors, and host cells encoding modified TdT polymerases, as well as methods including methods of producing modified TdT polymerases, and methods of using modified TdT polymerases (e.g., methods of nucleic acid synthesis).

Abstract: Methods are disclosed for high throughput identification of optimized template-independent polymerase variants. The methods comprise generation of polymerase gene-encoding variants, isolation of variants, expression of variants, exposing expressed polymerase variants to desired conditions for activity, and identification of active variants, such as by hybridization to synthesized polynucleotides or by amplification. Encoding genes of active variants can be sequenced. The method can be performed iteratively to enhance generation and selection of preferred variants.

Abstract: Disclosed herein is a method of utilizing an enzyme in a nucleic acid manipulation process, the method comprising: a) transforming a microorganism with a non-native enzyme; b) inducing expression of the enzyme in the microorganism, thereby producing the non-native enzyme; c) adding the microorganism of step b) directly to a non-naturally occurring nucleic acid manipulation process, wherein the non-native enzyme is not purified from the microorganism prior to addition to the nucleic acid manipulation process; and carrying out the nucleic acid manipulation process using the enzyme. Importantly, this method can be carried out without the need to purify the enzyme from the cell producing it before it is used in the nucleic acid manipulation method. Also disclosed herein is a kit for carrying out a nucleic acid manipulation process, the kit comprising a) a microorganism expressing a non-native enzyme; b) nucleic acids of interest; and c) reagents for use in the nucleic acid manipulation process.

Abstract: Embodiments of the disclosure concern methods and compositions related to generation and/or use of proofreading reverse transcriptases, including those that are thermophilic or hyperthermophilic. The disclosure encompasses specific recombinant polymerases and their use. In some embodiments, the polymerases are utilized for RNA sequencing in the absence of generation of a cDNA intermediate.

Abstract: Disclosed herein is a method of utilizing an enzyme in a nucleic acid manipulation process, the method comprising: a) transforming a microorganism with a non-native enzyme; b) inducing expression of the enzyme in the microorganism, thereby producing the non-native enzyme; c) adding the microorganism of step b) directly to a non-naturally occurring nucleic acid manipulation process, wherein the non-native enzyme is not purified from the microorganism prior to addition to the nucleic acid manipulation process; and carrying out the nucleic acid manipulation process using the enzyme. Importantly, this method can be carried out without the need to purify the enzyme from the cell producing it before it is used in the nucleic acid manipulation method. Also disclosed herein is a kit for carrying out a nucleic acid manipulation process, the kit comprising a) a microorganism expressing a non-native enzyme; b) nucleic acids of interest; and c) reagents for use in the nucleic acid manipulation process.

Abstract: Embodiments of the disclosure concern methods and compositions related to generation and/or use of proofreading reverse transcriptases, including those that are thermophilic or hyperthermophilic. The disclosure encompasses specific recombinant polymerases and their use. In some embodiments, the polymerases are utilized for RNA sequencing in the absence of generation of a cDNA intermediate.

Abstract: Disclosed herein is a method of utilizing an enzyme in a nucleic acid manipulation process, the method comprising: a) transforming a microorganism with a non-native enzyme; b) inducing expression of the enzyme in the microorganism, thereby producing the non-native enzyme; c) adding the microorganism of step b) directly to a non-naturally occurring nucleic acid manipulation process, wherein the non-native enzyme is not purified from the microorganism prior to addition to the nucleic acid manipulation process; and carrying out the nucleic acid manipulation process using the enzyme. Importantly, this method can be carried out without the need to purify the enzyme from the cell producing it before it is used in the nucleic acid manipulation method. Also disclosed herein is a kit for carrying out a nucleic acid manipulation process, the kit comprising a) a microorganism expressing a non-native enzyme; b) nucleic acids of interest; and c) reagents for use in the nucleic acid manipulation process.

Abstract: Embodiments of the disclosure concern methods and compositions related to generation and/or use of proofreading reverse transcriptases, including those that are thermophilic or hyperthermophilic. The disclosure encompasses specific recombinant polymerases and their use. In some embodiments, the polymerases are utilized for RNA sequencing in the absence of generation of a cDNA intermediate.

Abstract: Embodiments of the disclosure concern methods and compositions related to generation and/or use of proofreading reverse transcriptases, including those that are thermophilic or hyperthermophilic. The disclosure encompasses specific recombinant polymerases and their use. In some embodiments, the polymerases are utilized for RNA sequencing in the absence of generation of a cDNA intermediate.

Abstract: Embodiments of the disclosure concern methods and compositions related to generation and/or use of proofreading reverse transcriptases, including those that are thermophilic or hyperthermophilic. The disclosure encompasses specific recombinant polymerases and their use. In some embodiments, the polymerases are utilized for RNA sequencing in the absence of generation of a cDNA intermediate.

Abstract: Embodiments of the disclosure concern methods and compositions related to generation and/or use of proofreading reverse transcriptases, including those that are thermophilic or hyperthermophilic. The disclosure encompasses specific recombinant polymerases and their use. In some embodiments, the polymerases are utilized for RNA sequencing in the absence of generation of a cDNA intermediate.