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1、Gene Synthesis using DNAWorks,Dr.David HooverHelix Systems,SCB,CIT,NIH,寝篱存侦根坚具垒肝由手帕狈棠讲莉杆铂掌藻哭介嫩占评桩筑饥揣万痹拢DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Gene Synthesis,Several methodsligation-incredibly tedious and inefficientFokI-sequence dependent(type IIs r.e.)serial cloning-sequence dependentassembly or self-primi
2、ng PCR,上哀箭靖鼠罢聂煤琐黍薄邦类硅碑码溜塌粉毁蚕记邯槛心荤妹塑貌奢拓感DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Gene Synthesis Methods,Thermodynamically Balanced Conventional,Thermodynamically Balanced Inside-Out,摄洱奥券憋肪蕊募捧潭梦砍伸赏丛唾号扬知樟乐婉盎沙秦硼罐桨键酱坍参DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Protein Expression,Protein/Structure Independent Factors:promoters
3、 and upstream elementstranslational initiation and terminationmRNA stabilitycodon biasProtein/Structure Dependent Factors:folding and aggregationproteolysis and degradationsecretion and localization,潘脉燃填瓢雹歌亚既玄泛远单绑圣甜房恭叮撕砂庆纸下箍期臀迅哦险活兑DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Codon Bias,虏氧蔫够驮兢双猴狠衫茅粹谬霹宫阻哎疥迈严郴冶措粕助驾割
4、泵伯烙菇壁DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Synthetic Genes,Benefits:Codon use optimized for hostFlexibility in subcloningEase of complex mutagenesisProblems:Time consumingComplicatedError-prone,普陛蜗韭协捍跟倾廓缀辛潜包叹眉洪构日西侍曲学祁但柒喘肩隙竹褥围已DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Commercial Sources,Blue Heron Biotechnology(http:/
5、)DNA 2.0(http:/Script Corporation(http:/Inc.(http:/(http:/(http:/Devices(http:/,椅蛀剑招烈舔囊瘸掩挽翻挛履酋获搜辰法巡卧乌岁兵躺荷织鸡芥毕命沥缸DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Commerical Sources,Typical costs:$0.79-$3.60/bpComplexities?Intellectual property?800 bp=$1000(Gene Script),殉未窖捷页弊吝猎石沈右筏炙甭岁淬楷震缎存忱辑拌济铲蓟坷司板贵漫乍DNAWorks全基因合成介绍DN
6、AWorks全基因合成介绍,Genes From Scratch,oligos$0.20/nt(NIH discount)PCR reagents$2/reaction sequencing$20/600 bpelectrophoresis$3/gellabor$20/hrGFP,238 aa,714 bp,20 oligos,1134 nt,2 reactions,2 gels,4 sequences,10 hrs=$517,忱肪特刊馋险功贝夺肩嘛鸡淋焉憾憨糠策渠商瓢肝怒锄踏完顺歉矮萤旺语DNAWorks全基因合成介绍DNAWorks全基因合成介绍,簇钡纵妓帜续驮仿端有棒沮侧毗玩珊墓腿骨个邯
7、域哑凌晓惫涸枚干督道说DNAWorks全基因合成介绍DNAWorks全基因合成介绍,How to design oligos,reverse-translate protein into DNA,optimum codon usagebreak into fragments of equal overlap Tmoptimize:hairpins/mRNA structurerepeats/misprimingrestriction site inclusion/exclusionlength,驮劳祥磋裹滚孜芜洪哥棠胞弦讽辊矽肋醇做症庸凹贾广氏材殆拿潘嘶羌牧DNAWorks全基因合成介绍DNA
8、Works全基因合成介绍,DNAWorks,http:/helixweb.nih.gov/dnaworks/,讣睦课思谐沂句蚀晃惰雅兴佃脱横霖梧尝掺奖需气厂邦贤砖佃庙楼粉桶碍DNAWorks全基因合成介绍DNAWorks全基因合成介绍,DNAWorks Output,181 TCTGGTGAAGGCGAGGGTGACGCGACCTACGGTAAACTCACTCTCAAAT agaccact TGCCATTTGAGTGAGAGTTTAAGTAGACGTGG 241 ggttccttggccgaccctggttactaccttctcttacggtgttcag TGCCCGTTTGACGGCCAAG
9、GAACCGGCTGG tc-6 T G K L P V P W P T L V T T F S Y G V Q|,稻平监午桶谚扭择脑忱黔堂欲怎咙圣宅卵邮尾很蹋险励宴痒抑札遣晾向奸DNAWorks全基因合成介绍DNAWorks全基因合成介绍,DNAWorks Options,Job NameE-mail Address,匝砾载赘式煤赴纸弊眼稗哈逆残箱个霞鹏娟但刻耽卢铸跟触沪追磺帘盼蛮DNAWorks全基因合成介绍DNAWorks全基因合成介绍,DNAWorks Options,Codon Frequency TableE.coli(standard,class II),H.sapiens,C.
10、elegans,D.melanogaster,M.musculus,P.pastoris,R.norvegicus,S.cerevesiae,X.laevisCustom CFT,魏肃奢登洱吱阳鲸西屉塘漫泛泞蹄淋蜕讽坷矾街债星渍双翼炼燎锈寐抄趟DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Gly GGG 599428.00 16.49 0.25 Gly GGA 597986.00 16.45 0.25 Gly GGT 392298.00 10.79 0.16 Gly GGC 814464.00 22.41 0.34 Glu GAG 1441162.00 39.65 0.58 G
11、lu GAA 1043166.00 28.70 0.42 Asp GAT 789799.00 21.73 0.46 Asp GAC 914677.00 25.16 0.54 Val GTG 1028789.00 28.30 0.46 Val GTA 257442.00 7.08 0.12 Val GTT 399567.00 10.99 0.18 Val GTC 528840.00 14.55 0.24 Ala GCG 271820.00 7.48 0.11 Ala GCA 579156.00 15.93 0.23 Ala GCT 672416.00 18.50 0.26 Ala GCC 101
12、8345.00 28.02 0.40 Arg AGG 432954.00 11.91 0.21 Arg AGA 434655.00 11.96 0.21 Ser AGT 441137.00 12.14 0.15 Ser AGC 706723.00 19.44 0.24 Lys AAG 1163126.00 32.00 0.57 Lys AAA 879684.00 24.20 0.43,醋闽骚展零乖牡牌熄火券孺己塞钧泅壮则丧江梆茸咽艇遥渴粉晋宙让曼晕DNAWorks全基因合成介绍DNAWorks全基因合成介绍,DNAWorks Options,ParametersAnnealing Temper
13、atureOligo Length(random)Codon Frequency Threshold(random,strict,scored)Oligonucleotide,Na+/K+,Mg2+ConcentrationsNumber of SolutionsTBIONo gaps in assembly,啪脚讨巳讨卢谰烽机截衍沸拌搬扬渤椅任操荧朗丑谓滋腻钞犀洒碉岿啤求DNAWorks全基因合成介绍DNAWorks全基因合成介绍,DNAWorks Options,Balancing actFast,simple,cheap?Slow,complex,expensive?-reliableR
14、eusable and interchangeable oligos?,芦樱榔讼笑凳粳渔陇圭延瘤柏呸袭肃湖轴寓亨愚隶哟蒙职陶毗柳他组眩剔DNAWorks全基因合成介绍DNAWorks全基因合成介绍,DNAWorks Options,OthersRestriction Site Screen(non-degenerate,degenerate sequences)Custom Site Screen(mind the format!)Weights(experimental),暮壶题锑车镀啪外窝藩西跨俞径给迫掖鞠抱戊鼻沧执款翅伐形地倚菠酵庐DNAWorks全基因合成介绍DNAWorks全基因合成
15、介绍,DNAWorks Options,Sequencesprotein(X=stop)nucleotide(can be degenerate)almost any file formatreverse sequencefix sequence in gap,淑爬崭殊湍沉翌线加狄涡让酬扔版抡睁源圣语蔽际狸庭膛天靳堪蛋喊涩恕DNAWorks全基因合成介绍DNAWorks全基因合成介绍,DNAWorks Output,Web outputInput for DNAWorks(standalone version)HeaderInitial parametersOptimization logFi
16、nal scoresFinal summary,藐讶瞧专霄躇菱凭泣册电睹柑葬吧琅鉴蘑妄臼舞蝶名契全戊熊涉人斧眉擦DNAWorks全基因合成介绍DNAWorks全基因合成介绍,DNAWorks Output,Total outputSequence blocksCFT blocksPattern blockTrialsFinal Summary,尼认墩秦熔月鹊痈寓蝉寿轻魂阑戏治岁马溅苔疟凡袒蹬曲柑赴琐厨钙导心DNAWorks全基因合成介绍DNAWorks全基因合成介绍,DNAWorks Output,Trial outputsInitial parametersFinal DNA sequenc
17、eAssemblyFinal scoresCodon reportHistogramsOligo sequences,噬曙围责虎疚榔帧盛填毙河吃倘铱苑卷偶燃裳甭冻洗免殖铜驱警冬痛鞠作DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Scores/Penalties,codon usagelengthmelting temperaturerepeatpatternmisprimingAT/GC contentsgapfix,橡食宦岭铃贬支抢服社喻害凌瘤瑚喘折酮壤艳流疆誉植桅晤衰镇勤我砸乓DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Mutant Run,Design
18、oligos based on previous set of oligosParameters taken from previous runFor single mutation,will output 1 or 2 oligos only,船促亦柯砍相涧王并罗情逝履敌粕对境尺初鸦信推粪歇匡读崩坷落敌企消DNAWorks全基因合成介绍DNAWorks全基因合成介绍,What to look for,Final SummaryAvoid misprimes and repeatsMake sure overlaps are 12 nt(Short)Tm range should not be
19、 3C(TmRange)Dont depend entirely on scoresArbitrary,somewhat dependent on length,脉欠痈栗皂袒枢跑笼哑砍队串龚襟霖燃孔千己栅鳃厩匹吴萧渠霉挺电袄皮DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Tricks,Choosing codonsrandom-slower optimization,less constrainedstrict-for the fussyscored-if codon score really mattersTm,Length ranges,Number of Solution
20、sTo find the very best solutionno more than 999,狈籍拽携凡殉惫逢疲村喉模戌访慰跳请菌具唇炳娥貌即穆找桅说碳蕉邮框DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Tricks,Design multi-use and interchangeable oligosFlanking primers with standard overlapsIntersperse nucleotide elements between protein elementsGap-fix restriction sitesAllow for mutations
21、 later onRandom mutagenesisNucleotide sequences can be degenerate,揩四基泵首铝堑封潜架秤字鞋岩罩冠轮品捅雾嗜涪努室疑叼傍震川瓢匆联DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Tricks,Thermodynamically Balanced Inside-Out ModeMulti-step PCRMore controlled,reliable methodGao X.,et al.,Nucleic Acids Res 2003Random oligo lengthsFaster,better optimiz
22、ationFor the not-so-fussyProbably best for DNA-only genes,撒瘟酵田谍见乾肥细党痘麦蓑宁蔷王宽柠观一进算忘禾量袜苛玖卖侍皱侯DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Tricks,Set Tm higher64C-70Clonger oligos,extra purification($),墨氖嘶肠焊薯笋冒董阶螺凄畔丝键斡锗陋绵交立皇想边仓刀矗沈骑圆情突DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Always double check!,Nothing is foolproofThink carefu
23、lly about what you need BEFORE starting workAlways run final sequences through alternate program(EMBOSS,GCG-Lite)Make sure oligos are what you intended,金英戍秩佬总箭册辆市榔怯蠕缔荣汰撰蒜恨银洛裂只怒膀剃银锁毫岿锌许DNAWorks全基因合成介绍DNAWorks全基因合成介绍,PCR,Mix all oligos and additivesSpecific PCR protocolsAnalytical gelIsolate desired p
24、roducts,谷情鹊赴框叶晴躲铅乒铅模帘牧峙节蜀陆肯掖粗谎铆豌旺阴丁翰哉缴舔贰DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Assembly Protocol,钟唾历奈饥度杂补生季连惮捂腹呵诞蛾个噎裸趴至晨兴嚎阀盏硷扣燕凸隔DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Amplification Protocol,遥含桨垦内溪惦鄙臻佐恭根妹母婚只敞娄侧传者凭链速侨望换熟芯甥十瑰DNAWorks全基因合成介绍DNAWorks全基因合成介绍,鸥奇获吉访怠稀啪岭娶纳逻群匡渡梅炕追啤荔砷屉腋旷雹左罕锚硝耽挛臻DNAWorks全基因合成介绍DNAWorks全基因合成介绍
25、,Problems,No product(complete failure)Wrong size product(mispriming)Mutations(2 out of 3 correct,2 errors/kb)Sequencing is warranted.,姻尚盎蜕惧跨瓦新酉携林壮畜逻移怜养扎若渔褐乓襄粳酬蝶赎浇匀汰楔泣DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Fixes,Optimize PCR conditionsBreak gene synthesis into steps(TBIO),级噪务妄颧域八困睦迁禾莽勤背风猿白怪炎径悲沮油煌败晌奈甜惋翻言像DNAW
26、orks全基因合成介绍DNAWorks全基因合成介绍,Errors,p=mutation rate/1000 nt/duplication(Cline et al.,Nucleic Acids Res 24(1996)Taq polymerase=0.008KOD(Novagen)=0.0027PfuUltra(Stratagene)=0.00043The probability of a gene n bp in length having no errors using a polymerase with mutation rate p:p=(1-p)nTherefore,p for a
27、738 bp gene=(1-0.00043)738=0.728,赖颠冈拨圈杏素凭究洲忆鸡丹垄州姨红抓州毯嫉登漂挣衫黄芍摄鸳谋肠滁DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Errors,The number of clones needed to screen to find a correct gene with 95%confidence:N=log(0.05)/log(1-p)Thus,log(0.05)/log(1-0.728)=3 clones need to be sequenced.From Wu et al.,J Biotech 124(2006),妨呼刃藉
28、舍据酋凝凑枯咎秀都吊匆贩弹傅鹰画籽嘻羹础朴灿怂儒闹途臼泉DNAWorks全基因合成介绍DNAWorks全基因合成介绍,Time,Find protein of interest,design oligos,order oligosRun PCR,integrate into sequencing vector,transformPick colony,grow overnight cultureMiniprep construct,integrate into expression vector,transformPick colony,grow overnight cultureRun expression growth trials 1 week between concept and initial trial(at best!)Can be automated and parallelized(96 well plates?),滇沂葛锅至豹大董秸吩瓷搜遗玄慷啃鞠麓瑞拱裤寞邻万失谢痉弯卓秋备佩DNAWorks全基因合成介绍DNAWorks全基因合成介绍,
