苯丙酮酸羥化酶引物設計第1頁/共19頁第2頁/共19頁NCBIReferenceSequence:NM_000277.1

（美國國家生物技術中心參考序列：NM-000277.1）

CDS473..1831ORIGIN1cagctgggggtaaggggggcggattattcatataattgttataccagacggtcgcaggct61tagtccaattgcagagaactcgcttcccaggcttctgagagtcccggaagtgcctaaacc121tgtctaatcgacggggcttgggtggcccgtcgctccctggcttcttccctttacccaggg181cgggcagcgaagtggtgcctcctgcgtcccccacaccctccctcagcccctcccctccgg241cccgtcctgggcaggtgacctggagcatccggcaggctgccctggcctcctgcgtcagga301caagcccacgaggggcgttactgtgcggagatgcaccacgcaagagacaccctttgtaac361tctcttctcctccctagtgcgaggttaaaaccttcagccccacgtgctgtttgcaaacct421gcctgtacctgaggccctaaaaagccagagacctcactcccggggagccagcatgtccac481tgcggtcctggaaaacccaggcttgggcaggaaactctctgactttggacaggaaacaag541ctatattgaagacaactgcaatcaaaatggtgccatatcactgatcttctcactcaaaga601agaagttggtgcattggccaaagtattgcgcttatttgaggagaatgatgtaaacctgac661ccacattgaatctagaccttctcgtttaaagaaagatgagtatgaatttttcacccattt721ggataaacgtagcctgcctgctctgacaaacatcatcaagatcttgaggcatgacattgg781tgccactgtccatgagctttcacgagataagaagaaagacacagtgccctggttcccaag841aaccattcaagagctggacagatttgccaatcagattctcagctatggagcggaactgga901tgctgaccaccctggttttaaagatcctgtgtaccgtgcaagacggaagcagtttgctga第3頁/共19頁961cattgcctacaactaccgccatgggcagcccatccctcgagtggaatacatggaggaaga1021aaagaaaacatggggcacagtgttcaagactctgaagtccttgtataaaacccatgcttg1081ctatgagtacaatcacatttttccacttcttgaaaagtactgtggcttccatgaagataa1141cattccccagctggaagacgtttctcaattcctgcagacttgcactggtttccgcctccg1201acctgtggctggcctgctttcctctcgggatttcttgggtggcctggccttccgagtctt1261ccactgcacacagtacatcagacatggatccaagcccatgtatacccccgaacctgacat1321ctgccatgagctgttgggacatgtgcccttgttttcagatcgcagctttgcccagttttc1381ccaggaaattggccttgcctctctgggtgcacctgatgaatacattgaaaagctcgccac1441aatttactggtttactgtggagtttgggctctgcaaacaaggagactccataaaggcata1501tggtgctgggctcctgtcatcctttggtgaattacagtactgcttatcagagaagccaaa1561gcttctccccctggagctggagaagacagccatccaaaattacactgtcacggagttcca1621gcccctgtattacgtggcagagagttttaatgatgccaaggagaaagtaaggaactttgc1681tgccacaatacctcggcccttctcagttcgctacgacccatacacccaaaggattgaggt1741cttggacaatacccagcagcttaagattttggctgattccattaacagtgaaattggaat1801cctttgcagtgccctccagaaaataaagtaaagccatggacagaatgtggtctgtcagct1861gtgaatctgttgatggagatccaactatttctttcatcagaaaaagtccgaaaagcaaac第4頁/共19頁1921cttaatttgaaataacagccttaaatcctttacaagatggagaaacaacaaataagtcaa1981aataatctgaaatgacaggatatgagtacatactcaagagcataatggtaaatcttttgg2041ggtcatctttgatttagagatgataatcccatactctcaattgagttaaatcagtaatct2101gtcgcatttcatcaagattaattaaaatttgggacctgcttcattcaagcttcatatatg2161ctttgcagagaactcataaaggagcatataaggctaaatgtaaaacacaagactgtcatt2221agaattgaattattgggcttaatataaatcgtaacctatgaagtttattttctattttag2281ttaactatgattccaattactactttgttattgtacctaagtaaattttctttaggtcag2341aagcccattaaaatagttacaagcattgaacttctttagtattatattaatataaaaaca2401tttttgtatgttttattgtaatcataaatactgctgtataaggtaataaaactctgcacc2461taatccccataacttccagtatcattttccaattaattatcaagtctgttttgggaaaca2521ctttgaggacatttatgatgcagcagatgttgactaaaggcttggttggtagatattcag2581gaaatgttcactgaataaataagtaaatacattattgaaaagcaaatctgtataaatgtg2641aaatttttatttgtattagtaataaaacattagtagttta5’gaattcatgtccactgcggtcctgga（5'endprimer）（5’端引物）3’gtcgacttactgtattttctggaggg（3'endprimer）（3’端引物）第5頁/共19頁同源性檢驗Homologytest第6頁/共19頁同源性檢驗Homologytest同源序列·人類苯丙氨酸羥化酶·人類視網(wǎng)膜變性3（RD3），轉錄變異體2，mRNA·人類膜結合的轉錄因子的肽酶，位置2（MBTPS2），mRNA第7頁/共19頁Cleavagepointdetection酶切位點檢測第8頁/共19頁尋找引物的發(fā)夾結構searchingforhairpinstructuresofprimers第9頁/共19頁尋找互補的兩個引物配對searchingforcomplementarypairingoftwoprimers第10頁/共19頁尋找兩個引物的重復序列searchingforrepeatedmotifsoftwoprimers第11頁/共19頁分析引物的堿基和計算Tm值analysingbasedistributionofaprimerandcalculationofTmvalue第12頁/共19頁二.Carrierselectionandpreparation載體的選擇與準備Asthesizeofthetargetgeneislessthan10kb，sopreparepUC18/19plasmidvector。由于目的基因的大小小于10kb，所以準備pUC18/19質粒載體。第13頁/共19頁TheconnectionofthetargetDNAandthecarrier三．目的DNA與載體連接principle：usetheDNAligasetoconnect5'-phosphateand3`-OHwhichareclosedtoeachother原理：用DNA連接酶把相互靠近的5`-磷酸與3`-OH連到一起Selectstickyendconnectiondoubledigestedpointconnectionofthestickyends，fluorescentlabelingatthetheplasmidcleavagesites選擇黏端連接雙酶切點的粘性末端連接，在質粒的酶切位點處進行熒光標記第14頁/共19頁TherecombinantDNAintoahostcellforamplificationCalciumchlorideimport四．重組DNA轉入宿主細胞進行擴增氯化鈣導入法第15頁/共19頁ScreeningandIdentificationofrecombinant五．重組體的篩選與鑒定

Tactics：1）Firstscreenedcloningwithacarrier策略：1）先篩選出帶有載體的克隆Secondscreeningidentifiedcloningwithrecombinantvector2）然后篩選鑒定出帶有重組載體的克隆LastscreeningidentifiedcloningwiththepurposeofDNA3）最后篩選鑒定出帶有目的DNA的克隆第16頁/共19頁ScreeningandIdentificationofrecombinant五．重組體的篩選與鑒定

method：Antibioticresistancescreening方法：抗生素抗性篩選法第17頁/共19頁Detectionoftheexpressionofgenes六．基因表達的檢測WesternblotdetecttheexpressionofphenylalaninehydroxylaseWesternblot檢測苯丙氨酸羥化酶的表達WesternBlotusespolyacrylamidegelelectrophoresis，theobjecttobedetectedisprotein，antibodyistheprobe，usethemarkedsecondaryantibodytodisplaycolor。PAGEseparationofproteinsamples，Transfertoasolidphasecarrier(e.g.,cellulosenitratefilm)，Solidphasecarrieradsorpsproteinnon-covalently，andcankeepelectrophoreticseparationo