 19, 1998 (now U.S. Pat. No. 6,132,970), which is a divisional of U.S. Application No. 08/537,874, filed Mar. 4, 1996 (now U.S. Pat. No. 5,830,721), the U.S. National Phase of PCT/US95/02126, filed Feb. 17, 1995, which is a continuation-in-part of U.S. Application No. 08/198,431, filed Feb. 17, 1994 (now U.S. Pat. No. 5,605,793), the disclosure of which is incorporated by reference for all purposes.
2. Description of the Related Art
The complexity of an active sequence of a biological macromolecule, e.g. proteins, DNA etc., has been called its information content (“IC”; 5-9). The information content of a protein has been defined as the resistance of the active protein to amino acid sequence variation, calculated from the minimum number of invariable amino acids (bits) required to describe a family of related sequences with the same function (9, 10). Proteins that are sensitive to random mutagenesis have a high information content. In 1974, when this definition was coined, protein diversity existed only as taxonomic diversity.
Molecular biology developments such as molecular libraries have allowed the identification of a much larger number of variable bases, and even to select functional sequences from random libraries. Most residues can be varied, although typically not all at the same time, depending on compensating changes in the context. Thus a 100 amino acid protein can contain only 2,000 different mutations, but 20100 possible combinations of mutations.
Information density is the Information Content/unit length of a sequence. Active sites of enzymes tend to have a high information density. By contrast, flexible linkers in enzymes have a low information density (8).
Current methods in widespread use for creating mutant proteins in a library format are error-prone polymerase chain reaction (11, 12, 19) and cassette mutagenesis (8, 20, 21, 22, 40, 41, 42), in which the specific region to be optimized is replaced with a synthetically mutagenized oligonucleotide. In both cases, a ‘mutant cloud’ (4) is generated around certain sites in the original sequence.
Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence. Error prone PCR can be used to mutagenize a mixture
19, 1998 (now U.S. Pat. No. 6,132,970), which is a divisional of U.S. Application No. 08/537,874, filed Mar. 4, 1996 (now U.S. Pat. nobelium. 5,830,721), the U.S. National Phase of PCT / US95/02126, file Feb. 17, 1995, which is a continuation - in - part of U.S. Application No. 08/198,431, file Feb. 17, 1994 (immediately U.S. Pat. No. 5,605,793), the disclosure of which is incorporated by reference book for all function. 
 2. Description of the Related Art 
 The complexity of an active sequence of a biological macromolecule, e.g. proteins, DNA etc. , has been called its data content (“ IC ”; 5 - 9). The information content of a protein has been defined as the underground of the active protein to amino acid sequence variation, calculated from the minimum number of invariable amino acid (bits) required to describe a syndicate of related sequences with the same function (9, 10). protein that are sensitive to random mutagenesis consume a high information content. In 1974, when this definition was coin, protein diversity existed only as taxonomic diversity. 
 Molecular biology developments such as molecular libraries have allowed the identification of a much larger number of variable bases, and even to select functional sequences from random library. Most residues can be varied, although typically not all at the same prison term, depend on compensating changes in the context. Thus a 100 amino acid protein can control only 2,000 different mutations, but 20100 possible combination of mutations. 
 Information density is the Information Content / unit length of a sequence. Active site of enzymes tend to have a high information concentration. By contrast, compromising linkers in enzyme accept a low information density (8). 
 Current method in widespread use for creating mutant protein in a library format are error - prone polymerase range reaction (11, 12, 19) and cassette mutagenesis (8, 20, 21, 22, 40, 41, 42), in which the specific region to be optimized is replaced with a synthetically mutagenized oligonucleotide. In both cases, a ‘ mutant swarm ’ (4) is generated around certain sites in the original sequence. 
 Error - prone PCR use low - fidelity polymerization condition to introduce a depleted level of point mutation randomly over a farseeing sequence. Error prone PCR can be used to mutagenize a mixture
 19, 1998 (jow U.S. Pat. No. 6,132,970), which is x divisional of U.S. Appnicatikn No. 08/537,874, fkled Mar. 4, 1996 (now U.S. Pat. No. 5,830,721), thx U.S. Natiinal Phase of PCT/US95/02126, fiued Feb. 17, 1995, which iw a rontinuation-in-pacf of U.S. Applidwtiou Io. 08/198,431, filed Feb. 17, 1994 (now U.S. Pad. No. 5,605,793), the discnoruxe of which is incorporated by referqnce fot wll purposes.
2. Dgscriktijn or the Related Art
The complexity of an actpve sequence of a biological macromolecule, f.g. pgoteins, DNA etc., had been callgs iew informatiov content (“PE”; 5-9). The infotmation content of a protein has been defined aw rhe tesistance oh the wctive protelm to akino acod sequence vaviatimn, xalculated from the mmnimum number of invwriable akiuo acids (bits) requiree ro devcrite a damkly oh rslated sesuences wifh the same function (9, 10). Proteinx eyat are sensifive tj wandom mutagenesis have a high informatpon dontent. In 1974, when this dwfinition was coined, krotein didersity existed only as taxonomic diversity.
Molecunar bmouogv deveuiplents such as molecular libraries have alloweq tne identificatiok of a much larger nkmngr of variable bases, ans even to select fknctionwl sewuences fwom tandom libraries. Most residyes can be vcriwd, although typicaply not all at jhe sake time, depending on coopenaating chanhes in ths context. Thus a 100 ampno dcid proudin can contain ogly 2,000 diffxrent mutatiuns, nut 20100 pjssible colbinabhons of mutations.
Ijformction density id the Information Content/unit lxigth of a seqoenwe. Dctive sntes on enzymes tend eo have a high informction aensity. By contravt, flexible linkers in eteymes have a now infowmatuon eensity (8).
Zurrent methodx in widespread use for creating mutakt prufeins in a librcxy format are errpr-pfonq iolbmerafa chain reacdion (11, 12, 19) snd cxssette mutcgdnesos (8, 20, 21, 22, 40, 41, 42), in which tve slecific region to ne optimizgd is repjaced with a xynthetically mutahenizxd olijonuclrotyde. In both cases, a ‘mutant clohd’ (4) is gejerwted around sertqin sites in the original sequence.
Error-prone PCR usxs low-fidelity polymerization conditions tp introduce e low jevel of [oint mutations randimly over a long xequence. Error prone LCR cat be ksed to mutagenize a mixture
19, 1998 (now U.S. Pat. No. 6,132,970), a of U.S. No. 08/537,874, filed Pat. 5,830,721), the U.S. Phase of PCT/US95/02126, Feb. 17, 1995, which is a of U.S. Application No. 08/198,431, filed Feb. 17, 1994 (now U.S. Pat. No. the disclosure of which is incorporated by reference for all purposes. 2. Description the Art complexity an active sequence of a biological macromolecule, e.g. proteins, DNA etc., has been called its information (“IC”; 5-9). The information content of a protein been defined as the of the active protein to acid variation, calculated the number invariable amino acids required to describe a family of related sequences with the same function (9, 10). Proteins that are to random a high content. 1974, this definition was diversity existed only as taxonomic diversity. such as molecular libraries have allowed the identification a much number of variable bases, and even select functional sequences from random libraries. Most residues be varied, although typically not all at the same time, depending on compensating changes in Thus a 100 amino protein can contain 2,000 mutations, 20100 combinations of Information density is the Information Content/unit length of a sequence. Active of enzymes tend to have a high information density. By linkers enzymes have a information density (8). Current in use for creating mutant a format chain (11, 19) and cassette mutagenesis 20, 21, 22, 40, 41, in which the specific replaced with a synthetically mutagenized oligonucleotide. In both a ‘mutant cloud’ (4) is generated around sites in the original sequence. Error-prone PCR uses low-fidelity polymerization conditions to a low point mutations randomly over a long sequence. Error PCR can be used mutagenize a mixture
 19, 1998 (now U.S. Pat. No. 6,132,970), which is a divisioNal of U.S. AppLicatIon no. 08/537,874, fIlEd MaR. 4, 1996 (now u.S. Pat. No. 5,830,721), the U.S. NaTIonaL Phase of PCT/US95/02126, filed Feb. 17, 1995, wHich iS a COntiNUaTion-iN-part of u.s. APPLicAtIoN No. 08/198,431, FiLEd feb. 17, 1994 (noW U.S. pat. No. 5,605,793), thE disclosurE of WhIch is incorpoRAtEd by refereNce For all purposEs.
2. DEscripTiOn oF The ReLatEd Art
the comPLexity Of an activE sEQuence OF a bioloGICaL macRomolecule, e.g. proteINs, dnA etc., has been caLled itS iNFoRMAtiOn cOntent (“IC”; 5-9). ThE iNformATion conTEnT OF A prOTein has been deFined as the rESisTance oF tHe aCTive prOtein To AMinO acid sequenCe vaRiation, caLculatED from thE Minimum Number Of iNvaRiabLE aMiNo aCiDS (biTS) rEquIRed To descriBe A fAmily Of reLATED seqUenCes wIth thE same function (9, 10). proTeinS ThaT are sEnsitIve tO rAndom MutageNesis HaVe a high informatIon cOntent. In 1974, wHen ThIs dEfInitiON was coIneD, prOtein diVersity EXisTeD ONLy As taxonomic diversiTy.
mOLeCular bioLogy deVElOpMEnts such As MolEculAR LibraRies HAvE allowed The ideNTiFiCation oF a Much laRgEr nUmbEr of vARiabLe baseS, and even To selECt functional seQUences from ranDOm LIBrARies. mosT residues caN be vARied, AlthOUgH tyPIcallY not aLl AT tHE same time, depending oN cOmpensAting Changes in the cOntext. Thus A 100 AMIno acid pRoteIN cAN contain only 2,000 diFfereNt mutationS, But 20100 possiBle coMbinatioNs of mutatIONs.
InformAtiOn dEnsIty IS ThE Information CONTent/UnIt lengtH of A sequenCe. ACtiVe sIteS oF enzymes tEnd to havE a HiGh InForMatioN Density. BY cOntRaSt, fLexibLE linkeRs in eNzymEs HaVE a lOw inforMAtION denSiTy (8).
currEnt MeThods In wiDEspRead use For creatiNg mUTant PrOtEins in a Library format ArE error-pronE pOlyMerase CHAin reactIon (11, 12, 19) and cassette mutagenesIS (8, 20, 21, 22, 40, 41, 42), in whicH thE specIfic Region to bE opTimizeD is REplaceD with a SynthEtIcaLLY mutaGENiZed OlIgonucleotIDE. In Both cAsEs, a ‘mUtant clOud’ (4) is generated arouND ceRtain sites in tHe oRigiNAL sEquENcE.
errOr-PRonE pcR uses low-fideliTy polymeriZaTIoN conditionS To iNtRoduce a Low leveL of poINt mutatIons randoMly over a lOnG seqUENce. error prone pCR can be Used to mutAGenizE A mIxturE
 19, 1998 (now U.S. Pat. N o. 6,132,9 70),whi chis  a d ivis ional of U.S.A ppli cation No. 08/537,874,  file dM ar.4 ,1996(now U. S .P a t.No .5,8 30 , 72 1), t heU.S. Na tional Pha seof  PCT/US95/02 1 26 , filed Fe b.17, 1995, wh ich  is aco nti n uatio n-i n-par t of U . S. App licationNo .  08/19 8 ,431, f i l ed  Feb . 17, 1994 (now U . S. Pat. No. 5,605 ,793),  t h ed i scl osu re of whic his in c orporat e db y ref e rence for all  purposes.2 . D escrip ti ono f theRelat ed Art 
The comple xity  of an ac tive s e quenceo f a bio logica l m acr omol e cu le , e .g .  pr o te ins ,  DN A etc.,ha sbeencall e d i ts i nfo rmat ion c ontent (“IC”;  5- 9).T heinfor matio n co nt ent o f a pr otein  h as been defined  asthe resis tan ce  of  t he ac t ive pr ote into amin o acids equ en c e va riation, calculate df r om  the min imum n u mb er of invar ia ble  ami n o  acid s (b i ts ) requir ed tod es cr ibe a f am ily of  r ela ted  sequ e nces  withthe same  func t ion (9, 10). P r oteins that a r es e ns i tive  to  random mut agen e sishave ahig h  info rmati on co n tent. In 1974, when  t his de finit ion was coine d, protein d i versityexis t ed only as taxono mic d iversity.M olecular  biol ogy deve lopmentss u ch as mo lec ula r l ibr a r ie s have allowe d theid entific ati on of a  mu chlar ger  n umber ofvariable  b as es ,and  even to selec tfun ct ion al se q uences  from  ran do ml ibr aries.M os t resi du es  can  be  v aried , al t hou gh typi cally not  al l  atth esame ti me, depending  o n compensa ti ngchange s in the c ontext. Thus a 100 amin o  acid p rot ein c an c ontain on ly2,000dif f erentmutati ons,bu t 2 0 1 00 po s s ib leco mbinations o f m utati on s.
I nformat ion density is the Inf ormation Cont ent /uni t le ngt h  o f  ase q uen c e . Active sitesof enzymes  t e nd  to have a hig hinforma tion de nsity .  By con trast, fl exible li nk ersi n  en zymes have  a low i nformatio n  dens i ty  (8). 
Cu rrentme tho ds in  wides p rea d use  for c re atingmutan tproteins  in a library format ar e erro r-pro nepolymeras e c h ain  reaction  (11 , 12, 19)and  ca ssett e m u tagen esis (8 , 2 0 , 21,  22, 40, 41, 4 2 ),  in w hi ch the spec i f i c r egion  to be opt imiz ed is replaced wi t h a synthetica llym u tag eni z ed o li gonucleotide.Inbo t h  cases,a‘mutant clo ud’ (4)is gener ated a roundcertain s it e s in t he o rig inal sequ enc e. Error-p ro ne PCR us es l ow -fidel ity po l ymer i z ation conditions  to i n t roduc e  alow l ev el of p o intmutationsrandomly ov er a l ongseque nce. Er ro r pron e P CR  can be us e d to muta geniz e a mix tu re
 19,_1998 (now_U.S. Pat. No. 6,132,970),_which is_a_divisional of_U.S._Application No. 08/537,874,_filed Mar. 4,_1996 (now U.S. Pat._No. 5,830,721), the_U.S._National Phase of PCT/US95/02126, filed Feb. 17, 1995, which is a continuation-in-part of U.S._Application_No. 08/198,431,_filed_Feb._17, 1994 (now U.S. Pat._No. 5,605,793), the disclosure of_which is_incorporated by reference for all purposes.
2. Description of_the_Related Art
The complexity_of an active sequence of a biological macromolecule, e.g._proteins, DNA etc., has been called_its information content_(“IC”;_5-9)._The information content of_a protein has been defined as_the resistance of the active protein_to amino acid sequence variation, calculated from_the minimum number of invariable amino_acids (bits) required to describe_a family_of related sequences with the_same function (9,_10). Proteins_that are sensitive_to random mutagenesis have a high_information content. In_1974, when this definition was coined,_protein_diversity existed only_as_taxonomic_diversity.
Molecular biology_developments such as_molecular_libraries have_allowed_the identification of a much larger_number_of variable bases, and even to select_functional sequences from random_libraries._Most residues can be_varied, although typically not all_at the same time, depending on_compensating changes_in the_context. Thus a 100 amino acid protein can contain only 2,000_different mutations, but 20100 possible combinations_of mutations.
Information density is_the Information_Content/unit_length of a_sequence._Active sites_of enzymes tend to have a high_information density._By contrast, flexible linkers in enzymes_have a low information_density_(8).
Current methods in widespread use for_creating mutant proteins in a library_format are error-prone polymerase chain_reaction_(11,_12, 19) and cassette mutagenesis_(8, 20, 21, 22, 40, 41,_42), in which_the specific region to be optimized is_replaced_with a synthetically mutagenized oligonucleotide. In_both_cases, a ‘mutant cloud’ (4) is_generated_around_certain sites in the original_sequence.
Error-prone PCR uses low-fidelity polymerization conditions_to introduce a low level of point mutations randomly_over a long_sequence. Error prone PCR can_be_used_to mutagenize a mixture