Applied Biosystems, LLC

Applera Corporation

Trilink Biotechnologies, Inc.

Life Technologies Corporation

The Perkin-elmer Corporation

Gerald Zon

Victoria L Boyd

William A Andrus

Christie D McCollum

Alexandre Lebedev

Linda G Lee

Natasha Paul

Jonathan Shum

Sabrina Shore

MeriLisa Bozzini

Piotr J Guga

Provided herein are methods for ligation of polynucleotides containing modified ligation components, particularly modified ligase cofactors, modified acceptors and modified donors. The methods readily applied to ligation-based assays for detection of a nucleic acid sequence where the use of the modified cofactor improves discrimination between matched and mismatched templates. Furthermore, the use of the modified ligation components reduces or eliminates the ligation in the absence of nucleic acid template. In addition, methods are applied to the preparation of nucleic acid libraries using modified acceptor probes and modified donor probes that reduce or eliminate probe dimerization during the ligation process.

Chemically modified ligase cofactors, donors and acceptors

Provided herein are methods for ligation of polynucleotides containing modified ligation components, particularly modified ligase cofactors, modified acceptors and modified donors. The methods readily applied to ligation-based assays for detection of a nucleic acid sequence where the use of the modified cofactor improves discrimination between matched and mismatched templates. Furthermore, the use of the modified ligation components reduces or eliminates the ligation in the absence of nucleic acid template.

The present teachings provide methods, compositions, and kits for synthesizing and sequencing nucleic acids. In some embodiments, reversible di-nucleotide compounds are employed along with cleaving reactions that remove a label and a blocking moiety. Improved sequencing efficiency is achieved by the rapid polymerase-mediated incorporation of reversible di-nucleotide compounds. In some embodiments, the di-nucleotides do not contain conventional nucleotide triphosphates, but rather employ amino acid phosphoramidate nucleotides (AAPNs).

Reversible di-nucleotide terminator sequencing

The present invention provides methods and compositions for nucleic acid amplification. These methods involve the use of oligonucleotide primers in temperature dependent nucleic acid amplification reactions. In certain aspects, the methods are accomplished by use of certain modified oligonucleotide primers which provide utility in nucleic acid amplification. In preferred embodiments, the oligonucleotide primers are modified with particular chemical groups such as esters.

Phosphotriester-modified oligonucleotide primers for nucleic acid amplification

Compositions and methods for detecting 5-methylcytosine in a nucleic acid are disclosed. A 5-methylcytosine discriminator, which is a deoxyribonucleosidetriphosphate comprising a cytosine-pairing moiety such as a guanosine and a moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine is described. The discriminator is able to base pair with a cytosine but not a 5-methylcytosine. A 5-methylcytosine comprised by a target nucleotide can be detected in a reaction using a DNA polymerase and a primer hybridized immediately adjacent to the target nucleotide. In the reaction, pyrophosphate released upon incorporation of a dNTP complementary to a target nucleotide is detected. Lack of incorporation of the discriminator, but incorporation of a dGTP, can indicate that the target nucleotide is a 5-methylcytosine.

5-methylcytosine detection, compositions and methods therefor

Disclosed herein a reversible terminator nucleotides and methods of use.

Reversible terminator nucleotides and methods of use

The invention provides methods and materials for the conversion of cytosine to uracil. A nucleic acid, such a gDNA, is reacted with bisulfate, such as magnesium bisulfite, in the presence of a quaternary amine catalyst. Examples of suitable quaternary amine catalysts include but are not limited to quaternary ammonium compounds, quaternary alkyl ammonium salts, quaternary alkyl ammonium halides, quaternary methyl ammonium bromide, quaternary ammonium chloride, tetraethylammonium hydroxide, tetraethylammonium chloride, tetrabutylammonium chloride, tetrabutylammonium bromide. The invention also contemplates kits of premeasured ingredients for carrying out the methods of the invention either on an individual sample or on a plurality of samples.

Method and materials for quaternary amine catalyzed bisulfite conversion of cytosine to uracil

The invention provides methods and materials for conversion of cytosine to uracil. In some embodiments, a nucleic acid, such as gDNA, is reacted with at least one bisulfite salt having the formula XHSOor Y(HSO); wherein Xis ammonium ion, a tetraalkyl ammonium ion, or a group 1A ion other than sodium; and Yis a group 2A ion or a group 7B ion; under conditions effective to convert at least one cytosine nucleobase to a uracil nucleobase. In some embodiments, Xcomprises at least one of lithium ion, potassium ion, ammonium ion, tetraalkylammonium ion, magnesium ion, manganese ion and calcium ion. In some embodiments, the reacting is performed optionally in the presence of a polyamine catalyst and/or a quaternary amine catalyst. Also provided are kits that can be used to carry out methods of the invention.

Method and materials for bisulfite conversion of cytosine to uracil

The invention provides methods and materials for conversion of cytosine to uracil. In some embodiments, a nucleic acid, such as gDNA, is reacted with bisulfite and a polyamine catalyst, such as a triamine or tetra-amine. Optionally, the bisulfite comprises magnesium bisulfite. In other embodiments, a nucleic acid is reacted with magnesium bisulfite, optionally in the presence of a polyamine catalyst and/or a quaternary amine catalyst. Also provided are kits that can be used to carry out methods of the invention.

Method and materials for polyamine catalyzed bisulfite conversion of cytosine to uracil

The invention provides methods for purifying bisulfite-treated nucleic acid samples.

Bisulfite method

A method and system for polynucleotide synthesis are provided which employ solid phase synthesis on a nonswellable porous polystyrene support by phosphoramidite or hydrogen phosphonate chemistries. The polystyrene support gives rise to fewer tritylated failure sequences caused by chain growth from extraneous support sites, and allows lower amounts of monomer reactants to be used to achieve equal or better coupling efficiencies as those achieveable with CPG. The method and system also employ nucleoside intermediates whose exocyclic amines are protected by base-labile groups which permit simultaneous cleavage and deprotection of the completed polynucleotide chain in the presence of the solid phase support. This latter feature allows practical automation of both the synthesis and purification of polynucleotides.

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San Carlos, CA, United States of America

Gerald Zon