Applied Biosystems, LLC

The Perkin-elmer Corporation

Applera Corporation

Lynx Therapeutics, Inc.

Victoria L Boyd

Gerald Zon

David H Hawke

MeriLisa Bozzini

Pau-Miau Yuan

Karen L Fearon

Eugene G Spier

Timothy G Geiser

Robert J DeFranco

G Marc Loudon

Piotr J Guga

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

The invention relates generally to the field of nucleic acid sequence analysis. In certain embodiments, the analysis is genotyping. In certain embodiments, the analysis involves detecting single nucleotide polymorphisms (SNPs). The invention also relates to methods, kits, and computer software for nucleic acid analysis.

Methods and compositions for nucleotide analysis

A method of purifying a hydrophobically substituted polynucleotide by reverse phase HPLC is described. The hydrophobic substituent may be removed from the polynucleotide under non-acidic conditions; the purification method is thus especially useful for acid sensitive polynucleotide analogs.

Polynucleotide purification method

A solid support having surface-attached carboxylic acid groups is reacted with an isoxazolium salt to form an activated support having enol ester and sulfonate groups. After washing to remove residual isoxazolium salt and base, the activated support is dried. A polypeptide is covalently or non-covalently bound to the support. The resultant immobilized polypeptide can be conveniently sequenced by N- and C-terminal sequencing methods. The support can be a polyvinylidene difluoride membrane or a glass fiber membrane having surface attached carboxylic acid groups, and the enol and sulfonate groups are provided by reacting the support with 2-ethyl-5'-phenylisoxazolium sulfonate. The dry support can be stored for at least about 3 months to achieve a peptide immobilization yield that is substantially the same as the yield obtained in the absence of drying.

Immobilization of polypeptides on a polyvinylidene difluoride membrane

A method of forming a thiohydantoin from an N-protected amino acid is described. The method employs a phosphate compound selected from the group consisting of

Thiohydantoin formation and selective modification of the carboxy

A solid support and method for immobilization and sequence analysis of polypeptides. In one aspect, the invention is directed to a method for immobilizing a polypeptide on a solid support. In the method, a solid support having surface-attached carboxylic acid groups is reacted with an isoxazolium salt to form an activated support. After the activated support has been washed to remove residual isoxazolium salt and base, the support is dried. The dried support is then contacted with a polypeptide under conditions effective to bind the polypeptide covalently to the support. The immobilized polypeptide can be conveniently sequenced by N- and C-terminal sequencing methods.

Support and method for immobilizing polypeptides

A method is provided for C-terminal sequencing of a protein or peptide. An important feature of the method is the formation of an oxazolone moiety at the C-terminus of a protein or peptide by treatment with acetic anhydride under basic conditions followed by conversion of the oxazolone to a thiohydantoin moiety by treatment with thiocyanate under acidic conditions. Yields of thiohydantoin are further enhanced by delivering thiocyanate as the conjugate acid of a sterically hindered alkylammnonium cation.

Acetic anhydride activation for C-terminal protein sequencing

A method of forming a thiohydantoin from an N-protected amino acid. The method employs a uronium or phosphonium compound to activate the terminal carboxyl group of the amino acid and a thiocyanate reagent to cyclize the activated amino acid to the thio-hydantoin. The thiohydantoin can be cleaved from its N-protecting group, for use in C-terminal peptide sequencing. Particularly preferred uronium compounds include salts of 2-chlorouronium. Preferred thiocyanate reagents include trimethylsilyl isothiocyanate and crown ether adducts of metallothiocyanates, such as the 18-crown-6 adduct of KSCN.

Method of forming N-protected amino acid thiohydantoins

Disclosed is a method for enhancing the cleavage of an acyl thiohydantoin bond, for example, for use in C-terminal peptide sequencing. An acyl thiohydantoin is alkylated to form an adduct on the thiohydantoin, and the adduct-containing thiohydantoin is cleaved at its acyl bond by reaction with a cleaving agent under substantially anhydrous, acidic conditions. In C-terminal amino acid sequencing, the cleaved product is analyzed to identify the C-terminal amino acid.

Cleavage method for acyl thiohydantoins and use of the method in

A method of forming an amino acid thiohydantoin from an N-protected amino acid or the C-terminal amino acid of an N-protected peptide. The amino acid is activated by reaction with a ketenimine, and the activated ester is converted to the thiohydantoin by reaction with silyl or pyridine isothiocyanate. The ketenimine is generated by treating an N-substituted isoxazolium compound, such as Woodwards Reagent K with a base, preferably in the presence of the amino acid. Also disclosed is a solid phase support having a derivatized N-substituted isoxazolium or ketenimine group for use in the method.

Method for forming an amino acid thiohydantoin using an N-substituted

A method of C-terminal peptide sequencing. The peptide is reacted with a mixed anhydride of isothiocyanic acid and a carboxylic, carbonic, or sulfonic acid, under basic conditions, to produce a C-terminal peptidyl thiohydantoin. The C-terminal amino acid can be identified by cleaving the thiohydantoin from the peptide and identifying the free amino acid thiohydantoin.

Method of C-terminal peptide sequencing

A method of C-terminal peptide sequencing. The peptide is reacted with an activated support derivatized with a mixed anhydride of isothiocyanic acid and carboxylic or carbonic acid, under basic conditions. The peptidyl thiohydantoin which forms is separated from the solid support and further reacted with a cleaving agent carried on a second solid support, to release a free C-terminal amino acyl thiohydantoin from the peptide. The free thiohydantoin is analyzed to determine the C-terminal peptide residue. The residual peptide can be recycled through the supports for successive C-terminal residue determinations.

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San Carlos, CA, United States of America

Victoria L Boyd