Invitrogen Corporation

Life Technologies Corporation

Transgenomic, Inc.

Gary Floyd Gerard

Michael D Smith

Michael Leslie Kotewicz

Deb K Chatterjee

Robert Jason Potter

Kim Rosenthal

Gulshan Dhariwal

Paul D Taylor

Jun E Lee

Mekbib Astatke

Peter Qiu

Harini Shandilya

Reyes Candau

James M D'Alessio

Elizabeth Flynn

A-Li W Hu

The present invention is in the fields of molecular and cellular biology. The invention is generally related to reverse transcriptase enzymes and methods for the reverse transcription of nucleic acid molecules, especially messenger RNA molecules. Specifically, the invention relates to reverse transcriptase enzymes which have been mutated or modified to increase thermostability, decrease terminal deoxynucleotidyl transferase activity, and/or increase fidelity, and to methods of producing, amplifying or sequencing nucleic acid molecules (particularly cDNA molecules) using these reverse transcriptase enzymes or compositions. The invention also relates to nucleic acid molecules produced by these methods and to the use of such nucleic acid molecules to produce desired polypeptides. The invention also concerns kits comprising such enzymes or compositions.

Thermostable reverse transcriptases and uses thereof

The present invention relates to nucleic acid inhibitors, compositions and method for enhancing synthesis of nucleic acid molecules. In a preferred aspect, the invention relates to inhibition or control of nucleic acid synthesis, sequencing or amplification. Specifically, the present invention discloses nucleic acids having affinity for polypeptides with polymerase activity for use in such synthesis, amplification or sequencing reactions. The nucleic acid inhibitors are capable of inhibiting nonspecific nucleic acid synthesis under certain conditions (e.g., at ambient temperatures). Thus, in a preferred aspect, the invention relates to 'hot start' synthesis of nucleic acid molecules. Accordingly, the invention prevents, reduces or substantially reduces nonspecific nucleic acid synthesis. The invention also relates to kits for synthesizing, amplifying, reverse transcribing or sequencing nucleic acid molecules comprising one or more of the nucleic acid inhibitors or compositions of the invention. The invention also relates to using the inhibitors of the invention to prevent viral replication or treat viral infections in a subject. Thus, the invention relates to therapeutic methods and pharmaceutical compositions using the inhibitors of the invention. The invention thus may be used for in vivo and in vitro inhibition of nucleic acid synthesis and/or inhibition of polymerase activity.

Compositions and methods for temperature-dependent nucleic acid synthesis

The invention relates to methods for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule. The method involves cleaving a double-stranded nucleic acid molecule containing a mismatch with a mismatch-specific endonuclease which cleaves on the 3' side of the mismatch, and preserving the integrity of the variant nucleotide by ligating a double-stranded linker with a 3&#x2032;-overhang to said variant nucleotide. Because the variant nucleotide is immediately adjacent to the linker, PCR and/or sequence-by-synthesis analysis can be readily carried out.

Method for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule

The invention relates to methods for identifying the sequence of one or more variant nucleotides in nucleic acid molecules. The method involves cleaving a double-stranded nucleic acid molecule containing a mismatch with a mismatch-specific endonuclease which cleaves on the 3' side of the mismatch, and preserving the integrity of the variant nucleotide by ligating a Double-Stranded Linker with a degenerate 3&#x2032;-overhang to said variant nucleotide. Because the variant nucleotide is immediately adjacent to the linker, PCR and/or sequence-by-synthesis analysis can be readily carried out.

The present invention relates to isolated nucleic acid sequences encoding CEL II endonuclease and vectors and host cells for producing a protein encoded thereby.

Nucleic acid sequences encoding CEL II endonuclease

The present invention relates to the isolation and characterization of CEL I and CEL II endonuclease proteins. Methods and kits for identifying mismatches in double-stranded DNA are also provided.

Isolated CEL II endonuclease

A method of producing a Rous Sarcoma Virus reverse transcriptase (RSV RT) by expressing one orniore nucleic acid sequences encoding one or more subunits of RSV RT in a eukaryotic host cell and culturing the host cell under conditions sufficient to produce the recombinant RSV RT. The resulting RSV RT has a specific activity of between about 30,000 and 150,000 units per milligram and is suitable for methods including RT-polyinerase chain reaction (RT-PCR).

Recombinant method for making RSV reverse transcriptases and mutants thereof

The invention relates to compositions comprising mixtures of polypeptides having reverse transcriptase (RT) activity and to methods of producing, amplifying or sequencing nucleic acid molecules using these compositions or polypeptides, particularly at temperatures above about 55&#xb0; C. The invention also relates to nucleic acid molecules produced by these methods, to vectors and host cells comprising these nucleic acid molecules, and use of such nucleic acid molecules to produce desired polypeptide. The invention also relates to methods for producing Avian Sarcoma-Leukosis Virus (ASLV) RT subunits, in particular, Avian Myeloblastosis Virus (AMV) RTs, to isolated nucleic acid molecules encoding ASLV RT subunits, and to ASLV RT subunits produced by these methods. The invention further relates to nucleic acid molecules encoding recombinant RT holoenzymes, particularly ASLV RTs, methods for producing these RTs and to RTs produced by these methods. The invention also relates to kits comprising the compositions, polypeptides, and ASLV RTs of the invention.

Recombinant methods for making reverse transcriptases and mutants thereof

The present invention relates generally to compositions and methods for enhancing recombinational cloning of nucleic acid molecules. In particular, the invention relates to compositions comprising one or more ribosomal proteins and one or more additional protein components required for recombinational cloning. More particularly, the invention relates to such compositions wherein the ribosomal proteins are one or moreribosomal proteins, still more particularly wherein the ribosomal proteins are selected from the group ofribosomal proteins consisting of S10, S14, S15, S16, S17, S18, S19, S20, S21, L20, L21, and L23 through L34, and most particularly S20, L27, and S15. The invention also relates to the use of these compositions in methods for recombinational cloning of nucleic acids, in vitro and in vivo, to provide chimeric DNA molecules that have particular characteristics and/or DNA segments. The invention also relates to isolated nucleic acid molecules produced by the methods of the invention, to vectors comprising such nucleic acid molecules, and to host cells comprising such nucleic acid molecules and vectors.

Enhanced in vitro recombinational cloning of using ribosomal proteins

The invention relates to compositions comprising mixtures of polypeptides having reverse transcriptase (RT) activity and to methods of producing, amplifying or sequencing nucleic acid molecules using these compositions or polypeptides, particularly at temperatures above about 55&deg; C. The invention also relates to nucleic acid molecules produced by these methods, to vectors and host cells comprising these nucleic acid molecules, and use of such nucleic acid molecules to produce desired polypeptide. The invention also relates to methods for producing Avian Sarcoma-Leukosis Virus (ASLV) RT subunits, in particular, Avian Myeloblastosis Virus (AMV) RTs, to isolated nucleic acid molecules encoding ASLV RT subunits, and to ASLV RT subunits produced by these methods. The invention further relates to nucleic acid molecules encoding recombinant RT holoenzymes, particularly ASLV RTs, methods for producing these RTs and to RTs produced by these methods. The invention also relates to kits comprising the compositions, polypeptides, and ASLV RTs of the invention.

Composition of reverse transcriptases and mutants thereof

The present invention relates to nucleic acid inhibitors, compositions and method for enhancing synthesis of nucleic acid molecules. In a preferred aspect, the invention relates to inhibition or control of nucleic acid synthesis, sequencing or amplification. Specifically, the present invention discloses nucleic acids having affinity for polypeptides with polymerase activity for use in such synthesis, amplification or sequencing reactions. The nucleic acid inhibitors are capable of inhibiting nonspecific nucleic acid synthesis under certain conditions (e.g., at ambient temperatures). Thus, in a preferred aspect, the invention relates to &ldquo;hot start&rdquo; synthesis of nucleic acid molecules. Accordingly, the invention prevents, reduces or substantially reduces nonspecific nucleic acid synthesis. The invention also relates to kits for synthesizing, amplifying, reverse transcribing or sequencing nucleic acid molecules comprising one or more of the nucleic acid inhibitors or compositions of the invention. The invention also relates to using the inhibitors of the invention to prevent viral replication or treat viral infections in a subject. Thus, the invention relates to therapeutic methods and pharmaceutical compositions using the inhibitors of the invention. The invention thus may be used for in vivo and in vitro inhibition of nucleic acid synthesis and/or inhibition of polymerase activity.

Compositions and methods for enhanced sensitivity and specificity of nucleic acid synthesis

The invention relates to a gene which encodes reverse transcriptase having DNA polymerase activity and substantially no RNase H activity. The invention also relates to vectors containing the gene and hosts transformed with the vectors of the invention. The invention also relates to a method of producing reverse transcriptase having DNA polymerase activity and substantially no RNase H activity by expressing the reverse transcriptase genes of the present invention in a host. The invention also relates to a method of producing cDNA from mRNA using the reverse transcriptase of the invention. The invention also relates to a kit for the preparation of cDNA from mRNA comprising the reverse transcriptase of the invention.

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Frederick, MD, United States of America

Gary Floyd Gerard