Life Technologies Corporation

Invitrogen Corporation

Quanta Biosciences

Other

David M Schuster

Ayoub Rashtchian

Gulilat Gebeyehu

Joel A Jessee

Mark S Berninger

Wu-Bo Li

Jiulin Xia

George W Buchman, Iii

Methods for making eDNA molecules, for amplification of RNA by PCR and for preparation of cDNA libraries are provided. Kits for making eDNA molecules also are provided. Compositions are also provided comprising mixtures of reagents, including reverse transcriptases, buffers, cofactors and other components, suitable for immediate use in conversion of RNA into eDNA and RT PCR without dilution or addition of further components. These compositions are useful, alone or in the form of kits, for eDNA synthesis or nucleic acid amplification (e.g., by the Polymerase Chain Reaction) or for any procedure utilizing reverse transcriptases in a variety of research, medical, diagnostic, forensic and agricultural applications.

Compositions and methods for cDNA synthesis

Methods for making cDNA molecules, for amplification of RNA by PCR and for preparation of cDNA libraries are provided. Kits for making cDNA molecules also are provided. Compositions are also provided comprising mixtures of reagents, including reverse transcriptases, buffers, cofactors and other components, suitable for immediate use in conversion of RNA into cDNA and RT PCR without dilution or addition of further components. These compositions are useful, alone or in the form of kits, for cDNA synthesis or nucleic acid amplification (e.g., by the Polymerase Chain Reaction) or for any procedure utilizing reverse transcriptases in a variety of research, medical, diagnostic, forensic and agricultural applications.

The present invention provides improved methods for amplifying a nucleic acid molecule. More specifically, the invention provides methods for nucleic acid amplification which use primers having equivalent priming efficiency.

Use of predetermined nucleotides having altered base pairing characteristics in the amplification of nucleic acid molecules

The present invention is directed to compositions and methods for enhancing synthesis of nucleic acid molecules, particularly GC-rich nucleic acid molecules. Specifically, the invention provides compositions comprising one or more nitrogen-containing organic compounds having a formula selected from the group consisting of formula I and formula II (or salts or derivatives thereof), preferably 4-methylmorpholine N-oxide or betaine (carboxymethyltrimethylammonium), and further comprising one or more compounds selected from the group consisting of proline and an N-alkylimidazole compound, and more preferably proline, 1-methylimidazole or 4-methylimidazole. The invention further relates to methods for enhanced, high-fidelity synthesis of nucleic acid molecules, including via amplification (particularly PCR), reverse transcription, and sequencing methods. The invention also relates to nucleic acid molecules synthesized by these methods, to fragments or derivatives thereof, and to vectors and host cells comprising such nucleic acid molecules, fragments, or derivatives. The invention also relates to kits for synthesizing, amplifying, reverse transcribing or sequencing nucleic acid molecules comprising one or more of the compositions of the invention.

Methods for producing polypeptides through enhanced synthesis of encoding nucleic acid molecules

The present invention is directed to compositions and methods for enhancing synthesis of nucleic acid molecules, particularly GC-rich nucleic acid molecules. Specifically, the invention provides compositions comprising one or more nitrogen-containing organic compounds having a formula selected from the group consisting of formula I and formula II (or salts or derivatives thereof), preferably 4-methylmorpholine N-oxide or betaine (carboxymethyltrimethylammonium), and further comprising one or more compounds selected from the group consisting of proline and an N-alkylimidazole compound, and more preferably proline, 1-methyliimidazole or 4-methylimidazole. The invention further relates to methods for enhanced, high-fidelity synthesis of nucleic acid molecules, including via amplification (particularly PCR), reverse transcription, and sequencing methods. The invention also relates to nucleic acid molecules synthesized by these methods, to fragments or derivatives thereof, and to vectors and host cells comprising such nucleic acid molecules, fragments, or derivatives. The invention also relates to kits for synthesizing, amplifying, reverse transcribing or sequencing nucleic acid molecules comprising one or more of the compositions of the invention.

Compositions and methods for enhanced synthesis of nucleic acid molecules

The present invention provides improved methods, compositions and kits for amplifying a nucleic acid molecule. Specifically, the invention involves replacing at least one nucleotide of an oligonucleotide with nucleotide residue having altered base-pairing characteristics, such as inosine, hypoxanthine, xanthine, and methylated nucleotide derivatives, so as to more equalize the efficiency with which that oligonucleotide and a second oligonucleotide hybridize to a target molecule, and then amplifying the target molecule using, for example, the polymerase chain reaction. Improved amplification results from the improvement in the relative hybridization efficiencies.

Compositions, kits and uses of nucleotides having altered base pairing characteristics

The present invention provides improved methods for amplifying a nucleic acid molecule. More specifically, the invention involves replacing at least one nucleotide of an oligonucleotide with a nucleotide having altered base pairing characteristics, so as to more equalize the efficiency with which that oligonucleotide and a second oligonucleotide hybridize to a target molecule and then amplifying the target molecule using, for example, the polymerase chain reaction. Improved amplification results from the improvement in relative hybridization efficiencies.

Use of primers containing nucleotides having altered base pairing

The present invention provides improved methods for amplifying a nucleic acid molecule. Specifically, the invention involves replacing at least one nucleotide of an oligonucleotide with a deoxyinosine residue, so as to more equalize the efficiency with which that oligonucloetide and a second oligonucleotide hybridize to a target molecule, and then amplifying the target molecule using, for example, the polymerase chain reaction. Improved amplification results from the improvement in the relative hybridization efficiencies.

Use of deoxyinosine containing primers to balance primer efficiency in

A method and kit, employing exo-sample nucleotides such as deoxyuridine, capable of altering the nucleic acid sequence present at the 3' or 5' end of a DNA or RNA molecule is provided. The method and kit can be used to achieve the selective amplification of nucleic acid molecules.

Method for altering a nucleotide sequence

This invention discloses a scheme for producing nucleic acid end products that are functionally or exactly identical to the starting products, thereby resulting in exponential amplification of a desired nucleic acid sequence. Specifically, sequences are cycled between RNA and DNA forms using the following basic steps: (1) a T7 RNA polymerase promoter is ligated onto a single-stranded DNA template; (2) T7 RNA polymerase makes many copies of RNA: (3) a complementary DNA is made from the RNA by extension of a primer by reverse transcriptase; and (4) the RNA template is removed by ribonuclease H. This amplification method is useful for purposes such as genetic research and diagnostic assays.

Promoter ligation activated transcription amplification of nucleic acid

A method for amplifying a nucleic acid molecule which employs a proto-promoter-containing nucleic acid molecule having a blocked 3' terminus. The invention also includes kits containing reagents for conducting the method.

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Poolesville, MD, United States of America

David M Schuster