基因工程作業(yè)(引物設計)

基因工程作業(yè)基因工程作業(yè)10一、選擇緣由及應用口腔鱗癌組織中腫瘤轉(zhuǎn)移相關基因1〔MTA1〕在蛋白和mRNA的表達水平，提醒其與口腔鱗癌〔OSCC〕發(fā)生、進展的關系。方法承受免疫組織化學法和原位雜交技術檢測46OSCC標本、15例口腔黏膜白斑與20例正?？谇火つ吮局蠱TAl基因的表達水平，并分析其與OSCC臨床病理學參數(shù)的關系。結果MTA1蛋白和MTA1mRNA在OSCC〔P〈0．0，口腔黏膜白斑中MTA1蛋白和MTA1mRNA表達水平顯著高于口腔正常黏膜〈001MTA1蛋白和MTA1mRNA表達與腫瘤浸潤深度和淋巴結轉(zhuǎn)移親熱相關〔〈．05。結論MTA1基因在蛋白和mRNA的表達水平在OSCCOSCC預后及選擇治療方案的一個腫瘤標志物。二、2NCBI得到MTA1相關信息并獲得目的基因PREDICTED:Gorillagorillagorillametastasisassociated1(MTA1),mRNANCBIReferenceSequence:XM_004055801.1FASTAGraphicsLOCUS XM_004055801 2872bp mRNA linearPRI03-DEC-2023DEFINITIONPREDICTED:Gorillagorillagorillametastasisassociated1(MTA1),mRNA.ACCESSION XM_004055801VERSION XM_004055801.1GI:426378238KEYWORDS .SOURCE Gorillagorillagorilla(westernlowlandgorilla)ORGANISMGorillagorillagorillaEuteleostomi;Mammalia;Eutheria;Euarchontoglires;Primates;Haplorrhini;Catarrhini;Hominidae;Gorilla.COMMENT MODELREFSEQ:Thisrecordispredictedbyautomatedcomputationalsequence

analysis.Thisrecordisderivedfromagenomic4)dgemethod:GNOMON,supportedbymRNAandESTevidence.Alsosee:DocumentationofNCBI”sAnnotationProcessRelease100

##Genome-Annotation-Data-START##AnnotationProvider::NCBIAnnotationStatus ::FullannotationAnnotationVersion::GorillagorillaAnnotationAnnotationPipeline::NCBIeukaryoticgenomeannotationpipelineAnnotationMethod ::Best-placedRefSeq;GnomonFeaturesAnnotated::Gene;mRNA;CDS;ncRNA##Genome-Annotation-Data-END##FEATURES Location/Qualifierssource 1..2872/organism=“Gorillagorillagorilla“/mol_type=“mRNA“/sub_species=“gorilla“/db_xref=“tax9o5n9:5“/chromosome=“14“gene 1..2872/gene=“MTA1“/note=“Derivedbyautomatedcomputationalanalysisusingevidence7Proteins“

genepredictionmethod:GNOMON.Supportingincludessimilarityto:4mRNAs,303ESTs,依據(jù)misc_feature 104..106/gene=“MTA1“/note=“upstreamin-framestopcodon“CDS 212..2359/gene=“MTA1“/codon_start=1/product=“metastasis-associatedproteinMTA1“

/protein_iXdP=_“004055849.1“/db_xref=“GI:426378239“/db_xref=“Gene1I0D1:134898“/translation=“MAANMYRVGDYVYFENSSSNPYLIRRIEELNKTANGNVEAKVVCFYRRRDISSTLIALADKHATLSVCYKAGPGADNGEEGEIEEEMENPEMVDLPEKLKHQLRHRELFLSRQLESLPATHIRGKCSVTLLNETESLKSYLEREDFFFYSLVYDPQQKTLLADKGEIRVGNRYQADITDLLKEGEEDGRDQSKLETKVWEAHNPLTDKQIDQFLVVARSVGTFARALDCSSSVRQPSLHMSAAAASRDITLFHAMDTLHKNIYDISKAISALVPQGGPVLCRDEMEEWSASEANLFEEALEKYGKDFTDIQQDFLPWKSLTSIIEYYYMWKTTDRYVQQKRLKAAEAESKLKQVYIPNYNKPNPNQISVNNVKAGVVNGTGAPGQSPGAGRACESCYTTQSYQWYSWGPPNMQCRLCASCWTYWKKYGGLKMPTRLDGERPGPNRSNMSPHGLPARSSGSPKFAMKTRQAFYLHTTKLTRIARRLCREILRPWHAARHPYLPINSAAIKAECTARLPEASQSPLVLKQAVRKPLEAVLRYLETHPRPPKPDPVKSVSSVLSSLTPAKVAPVINNGSPTILGKRSYEQHNGVDGNMKKRLLMPSRGLANHGQTRHMGPSRNLLLNGKSYPTKVRLIRGGSLPPVKRRRMNWIDAPDDVFYMATEETRKIRKLLSSSETKRAARRPYKPIALRQSQALPLRPPPPAPVNDEPIVIED“ORIGIN1tcctcctcttcctctcccgcccgcgccgcggccctcccgtccctgcgcggcctcggcggc61ctcggcggcggcggcggcggcggcggcagcagcgcggcccctttaaacgcctgcggcgcc121ccccgcccccgccatcgcgcctccattttcccggccgcccgcgccgagcgccgcgcccgc 起始密碼子181cccgggcccctccgccgccgccggcccggacatggccgccaacatgtacagggtcggaga241ctacgtctactttgagaactcctccagcaacccatacctgatccggaggatcgaggagct301caacaagacggccaatgggaacgtggaggccaaagtggtgtgcttctaccggaggcggga361catctccagcaccctcatcgccctggccgacaagcacgcaaccctgtcagtctgctataa421ggccggaccgggggcggacaacggcgaggaaggggaaatagaagaggaaatggagaatcc481ggaaatggtggacctgcccgagaaactaaagcaccagctgcggcatcgggagctgttcct541ctcccggcagctggagtctctgcccgccacgcacatcaggggcaagtgcagcgtcaccct601gctcaacgagaccgagtcgctcaagtcctacctggagcgggaggatttcttcttctattc661tctagtctacgacccacagcagaagaccctgctggcagataaaggagagattcgagtagg721aaaccggtaccaggcagacatcaccgacttgttaaaagaaggcgaggaggatggccgaga781ccagtccaagttggagaccaaggtgtgggaggcgcacaacccactcacagacaagcagat841cgaccagttcctggtggtggcccgctctgtgggcaccttcgcacgggccctggactgcag901cagctccgtccgacagcccagcctgcacatgagcgccgcagctgcctcccgagacatcac961gctgttccacgccatggatactctccacaagaacatctatgacatctccaaggccatctc1021ggcactggtgccgcagggcgggcccgtgctctgcagggacgagatggaggagtggtctgc1081atcagaggccaaccttttcgaggaagccctggaaaaatatgggaaggatttcacggacat1141tcagcaagattttctcccgtggaagtcgctgaccagcatcattgagtactactacatgtg1201gaagaccaccgacagatacgtgcagcagaaacgcttgaaagcagctgaagctgagagcaa1261gttaaagcaagtttatattcccaactataacaagccaaatccgaaccaaatcagtgtcaa1321caacgtcaaggccggtgtggtgaatggcacgggggcgccgggccagagccctggggctgg1381ccgggcctgcgagagctgttacaccacacagtcttaccagtggtattcttggggtccccc1441taacatgcagtgtcgtctctgcgcatcttgttggacatattggaagaaatatggtggctt1501gaaaatgccaacccggttagatggagagaggccaggaccaaaccgcagtaacatgagtcc1561ccacggcctcccagcccggagcagcgggagccccaagtttgccatgaagaccaggcaggc1621tttctatctgcacacgacgaagctgacgcggatcgcccggcgcctgtgccgtgagatcct1681gcgcccgtggcacgctgcgcggcacccctacctgcccatcaacagtgcggccatcaaggc1741cgagtgcacggcgcggctgcccgaagcctcccagagcccgctggtgctgaagcaggcggt1801acgcaagccgctggaagccgtgcttcggtatcttgagacccacccccgtccccccaagcc1861tgaccccgtgaaaagcgtgtccagcgtgctcagcagcctgacgcccgccaaggtggcccc1921cgtcatcaacaacggctcccccaccatcctgggcaagcgcagctacgagcagcacaacgg1981ggtggacggcaacatgaagaagcgcctcttgatgcccagtaggggtctggcaaaccacgg2041acagaccaggcacatgggaccaagccggaacctcctgctcaacgggaagtcctaccccac2101caaagtgcgcctgatccgggggggctccctgcccccagtcaagcggcggcggatgaactg2161gatcgacgccccggatgacgtgttctacatggccacagaggagaccaggaagatccgcaa2221gctgctctcatcctcggaaaccaagcgtgctgcccgccggccctacaagcccatcgccct2281gcgccagagccaggccctgccgctgcggccaccgccacctgcgcccgtcaacgacgagcc 終止密碼子2341catcgtcatcgaggactaggggccgcccccacctgcggccgccccccgcccctcgcccgc2401ccacacggccccttcccagccagcccgccgcccgcccctcagtttggtagtgccccacct2461cccgccctcacctgcagagaaacgcgctccttggcggacactgagggaggagaggaagaa2521gcgcggctaacttattccgagaatgccgaggagttgtcgtttttagctttgtgtttactt2581tttggctggagcggagatgaggggccaccccgtgcccctgtgctgcggggccttttgccc2641ggaggccgggccctaaggttttgttgtgttctgttgaaggtgccgttttaaattttattt2701ttattactttttttgtagatgaacttgagctctgtaacttacacctggaatgttaggatc2761gtgcggccgcggccggccgagctgcctggcggggttggcccttgtcttttcaagtaattt2821tcatattaaacaaaaacaaagaaaaaaatcttataaaaaggaaaaaaaccaa//三、表達載體的選擇我所選用的原核表達載體為質(zhì)粒pBR322；pBR322是一種常用的E.coli克隆載體〔，為4,361bp的環(huán)狀雙鏈DN〔pBR322攜帶有氨芐青霉素和四環(huán)素的抗性基因，且能在氯霉素存在條件下增殖。四、宿主菌為大腸桿菌。五、酶切位點primer5.0檢查編碼序列經(jīng)檢驗編碼序列中沒有EcoRIHindIIIEcoRIHindIII識別位點 為了使目的基因片段能連接到載體質(zhì)粒pBR322上，所以在目的片段的5’端和3’端分別連接上HindⅢ識別序列和EcoRI識別序列，如下5’——G↓AATTCATGGCCGCCAACATGTACA……CATCGTCATCGAGGACTAGA↓AGCTT 3’3’----CTTAA↑GTACCGGCGGTTGTACATGT……GTAGCAGTAGCTCCTGATCTTCGA↑A 5’↓ ↓EcoRI識別位點 HindIII識別位點六、引物設計PCR引物設計原理：PCR引物設計的目的是為了找到一對適宜的核苷酸片段，使其能有效地擴增模板DNA序列。PCR引物設計原則：15-30bp18-27bp38，由于過長會導74TaqDNA聚合酶進展反響。引物序列在模板內(nèi)應當沒有相像性較高，尤其是3’端相像性較高的序列，否則簡潔導致錯配。引物3’3個以上的連續(xù)堿基，如GGGCCC，也會使錯誤引發(fā)機率增加。GC含量不能相差太大。DNAPCR產(chǎn)物的載體的相應序列而確定。這種狀況下，設計引物要長一些。第一部：擴增根本序列5’——ATGGCCGCCAACATGTACA……CATCGTCATCGAGGACTAG 3’……GTAGCAGTAGCTCCTGATC 5’primer23’----TACCGGCGGTTGTACATGT……GTAGCAGTAGCTCCTGATC 5’5’------ATGGCCGCCAACATGTACA primer1其次步：GC比值和Tm值Primer15’------ATGGCCGCCAACATGTACA……(GC:53%Tm=58)Primer25’----CTAGTCCTCGATGACGATG----(GC:53%Tm=58)Primer15’ ATGGCCGCCAACATGTACA(GC:53%Tm=58)Primer25’ CTAGTCCTCGATGACGATG第三步：酶切位點(GC:53%Tm=58)Primer15’ GAATTCATGGCCGCCAACATGTACAPrimer25’ AAGCTTCTAGTCCTCGATGACGATG第四步：保護序列Primer15’ GCGCGAATTCATGGCCGCCAACATGTACAPrimer25’ GCGGAAGCTTCTAGTCCTCGATGACGATG此為保護序列故得出結論：1EcoRI：2HindIIIPrimer15’ GCGCGAATTCATGGCCGCCAACATGTACA基因工程作業(yè)基因工程作業(yè)11Primer25’ GCGGAAGCTTCTAGTCCTCGATGACGATG七、過程1、PCR體外擴增反響體系：模板0.5ul上游引物1ul下游引物1ul10XTaqDNA聚合酶緩沖液2.5uldNTPf2．5mM)2ulTaq酶1uldH2017ul總量25ul程序設計1、94℃預變性 2min2、94℃變性 45s3、58℃退火 30s4、70℃延長 30s52—4,30次6、72℃延長 3