Patent Application: US-65593110-A

Abstract:
this patent applications concerns compositions of matter that are dna polymerases , where those polymerases have had one or more of their amino acids replaced at sites chosen by an analysis of patterns of conservation and replacement within homologous protein sequences . disclosed here are sites within family a dna polymerases where amino acid replacement creates polymerases having utility , in an example where dna nucleotides are incorporated having modified or unnatural nucleobases , and / or nucleotides whose sugar is unnatural or derivatized , including 3 ′- o - amino - 2 ′- deoxyribonucleoside triphosphates . the claimed compositions include polymerases that hold amino acid replacements at claimed sites in taq polymerase , and are prepared by site - directed mutagenesis that modifies a gene encoding a parent taq gene to change the codon encoding the amino acid at the claimed site , giving a variant gene that encodes a taq polymerase protein that has a different amino acid at the claimed site .

Description:
the dna polymerase from thermus aquaticus ( taq ) is commonly used for sanger - based sequencing methods and it is known to release primer / template complexes . unfortunately , wild - type taq polymerase may not be able to incorporate t - onh 2 well enough to support the sequencing - by - synthesis method outlined above , or other versions of it . polymerases that have had amino acid replacement ( s ) ( one or more ) so that the variant polymerase ( defined here to be different from the parent polymerase , which may be the same or different from a polymerase found in nature ) that accept triphosphates modified to carry reversibly terminating units ( on the sugars as well as elsewhere , such as on the nucleobases ) will therefore have utility . a process to construct useful variants , or the novel compositions that those variants are , must begin with a process that identifies sites in a polymerase protein sequence where the amino acid is changed . a “ site ” is , as defined here ”, specified by a number in a sequence of amino acids , where that number is defined by reference to a figure that lists the amino acids of the protein in that sequence . disclosed elsewhere ( e . g . u . s . pat . no . 5 , 958 , 784 ) is a semi - rational approach , defined here as a “ phylogenetic - based approach ”, which begins by the process of analyzing the patterns of change and conservation among a set of homologous proteins . this process comprises some or all of the following steps : constructing evolutionary trees from the multiple sequence alignment , inferring the sequences of ancestral proteins at nodes in the tree , applying phylogenetic tools to identify signals / sites associated with functional divergence , and examining these sites together with a model representing the three dimensional structure of the protein . this approach identifies a small number of sites where amino acid replacement might yield a useful protein , specifically , one that is not catalytically active or that does not fold properly . thus , this process complements other directed evolution approached . to apply this process to dna polymerases that are members of evolutionary family a , the sequences of 719 polymerases from family a were collected from the pfam database ( pf00476 ) [ bat04 ]. a phylogenetic tree representing the evolutionary relationship of these polymerases was obtained from the pfam database ( pf00476 ) [ bat04 ]. metrics used to infer functional divergence were applied to the dataset . this computational phylogenetic - based approach confirmed what was already proposed in the literature : functional divergence of polymerase behaviors has occurred along branches of the phylogeny separating viral and non - viral polymerases [ hor95 ][ lea06 ][ sis06 ][ tab95 ]. owing to the fact that viral polymerases are inherently more likely to accept modified nucleotides than non - viral polymerases , the phylogenetic - based approach implied that sites identified as being responsible for functional divergence between viral and non - viral polymerases would be sites that , in the non - viral polymerases , could have their amino acids replaced to generate new polymerase variants with useful properties . making reference to a model for the three dimensional crystal structure of polymerases , phylogenetic - based analysis identified sites both within and without the active - site cleft of the polymerase . rationally , sites within the active site are more likely to alter substrate specificity [ hen05 ]. amino acid replacements distributed across 35 of these sites were identified as having potential interest . according to the process of the invention , one of these sites may be changed , or more than one of these sites may be changed , to generate a useful polymerase . further , these sites may be changed starting with a polymerase parent from any member of family a ( either as found naturally or as found from a laboratory that has already one or more amino acids different from a natural family a polymerase . the taq polymerase protein is widely used for sequencing dna . standard experimental approaches are used to perform site - directed mutagenesis to incorporate the amino acid replacements at sites listed in the claims . for instance , mutagenic pcr is performed on a template family a polymerase gene using the appropriate mutagenic primers to generate variants containing amino acid replacements . the pcr mixtures contain the following : 1 × mutagenic taq buffer ( 10 mm tris - hcl , ph 8 . 3 , 50 mm kcl , 15 mm mgcl 2 ), 0 . 1 ng / μl template dna , 200 μm dntps , 300 nm p - 4 , 300 nm mutagenic primers , 5 u taq polymerase ( new england biolabs , beverly , mass . ), and mgcl 2 . pcr reaction continues as follows : 5 min , 94 ° c . ; ( 30 s , 94 . 0 ° c . ; 20 s , 55 . 0 ° c . ; 3 min , 72 . 0 ° c . )× 15 cycles ; 7 min , 72 . 0 ° c . ; 4 . 0 ° c . products can purified with the qiaquick pcr purification kit ( qiagen , valencia , calif . ), eluted with qiagen buffer eb ( 50 μl ), and quantitated at an absorbance of 260 nm using a spectrophotometer . this invention provides new sequences of proteins that are likely to accept nucleoside 3 ′- onh 2 blocked triphosphates , dideoxynucleoside triphosphates and c - glycosides such as 2 ′- deoxypseudouridine - 5 ′- triphosphate . 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