摘要:
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.
摘要:
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.
摘要:
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.
摘要:
A method and system for polynucleotide synthesis and purification 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 squences caused by chain growth from extraneous support sites. Consequently, currently used rapid purification techniques depending on trityl hydrophobicity give a more highly purified product. 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.
摘要:
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).
摘要:
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.
摘要:
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.
摘要:
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, tetraethyl ammonium hydroxide, tetraethylammonium chloride, tetrabutyl ammonium chloride, tetrabutyl ammonium 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.