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1、,分子生物学,Homo sapiens phenylalanine hydroxylase (PAH), mRNA,苯丙氨酸羟化酶,http:/www.ppthi-,NCBI Reference Sequence: NM_000277.1 （美国国家生物技术中心参考序列：NM-000277.1） CDS 4731831 ORIGIN,http:/www.ppthi-,1 cagctggggg taaggggggc ggattattca tataattgtt ataccagacg gtcgcaggct61 tagtccaatt gcagagaact cgcttcccag gcttctgaga g
2、tcccggaag tgcctaaacc121 tgtctaatcg acggggcttg ggtggcccgt cgctccctgg cttcttccct ttacccaggg181 cgggcagcga agtggtgcct cctgcgtccc ccacaccctc cctcagcccc tcccctccgg241 cccgtcctgg gcaggtgacc tggagcatcc ggcaggctgc cctggcctcc tgcgtcagga301 caagcccacg aggggcgtta ctgtgcggag atgcaccacg caagagacac cctttgtaac361
3、tctcttctcc tccctagtgc gaggttaaaa ccttcagccc cacgtgctgt ttgcaaacct421 gcctgtacct gaggccctaa aaagccagag acctcactcc cggggagcca gcatgtccac481 tgcggtcctg gaaaacccag gcttgggcag gaaactctct gactttggac aggaaacaag541 ctatattgaa gacaactgca atcaaaatgg tgccatatca ctgatcttct cactcaaaga601 agaagttggt gcattggcca aa
4、gtattgcg cttatttgag gagaatgatg taaacctgac661 ccacattgaa tctagacctt ctcgtttaaa gaaagatgag tatgaatttt tcacccattt721 ggataaacgt agcctgcctg ctctgacaaa catcatcaag atcttgaggc atgacattgg781 tgccactgtc catgagcttt cacgagataa gaagaaagac acagtgccct ggttcccaag841 aaccattcaa gagctggaca gatttgccaa tcagattctc agct
5、atggag cggaactgga901 tgctgaccac cctggtttta aagatcctgt gtaccgtgca agacggaagc agtttgctga,http:/www.ppthi-,961 cattgcctac aactaccgcc atgggcagcc catccctcga gtggaataca tggaggaaga1021 aaagaaaaca tggggcacag tgttcaagac tctgaagtcc ttgtataaaa cccatgcttg1081 ctatgagtac aatcacattt ttccacttct tgaaaagtac tgtggctt
6、cc atgaagataa1141 cattccccag ctggaagacg tttctcaatt cctgcagact tgcactggtt tccgcctccg1201 acctgtggct ggcctgcttt cctctcggga tttcttgggt ggcctggcct tccgagtctt1261 ccactgcaca cagtacatca gacatggatc caagcccatg tatacccccg aacctgacat1321 ctgccatgag ctgttgggac atgtgccctt gttttcagat cgcagctttg cccagttttc1381 cc
7、aggaaatt ggccttgcct ctctgggtgc acctgatgaa tacattgaaa agctcgccac1441 aatttactgg tttactgtgg agtttgggct ctgcaaacaa ggagactcca taaaggcata1501 tggtgctggg ctcctgtcat cctttggtga attacagtac tgcttatcag agaagccaaa1561 gcttctcccc ctggagctgg agaagacagc catccaaaat tacactgtca cggagttcca1621 gcccctgtat tacgtggcag
8、agagttttaa tgatgccaag gagaaagtaa ggaactttgc1681 tgccacaata cctcggccct tctcagttcg ctacgaccca tacacccaaa ggattgaggt1741 cttggacaat acccagcagc ttaagatttt ggctgattcc attaacagtg aaattggaat1801 cctttgcagt gccctccaga aaataaagta aagccatgga cagaatgtgg tctgtcagct1861 gtgaatctgt tgatggagat ccaactattt ctttcatca
9、g aaaaagtccg aaaagcaaac,http:/www.ppthi-,1921 cttaatttga aataacagcc ttaaatcctt tacaagatgg agaaacaaca aataagtcaa1981 aataatctga aatgacagga tatgagtaca tactcaagag cataatggta aatcttttgg2041 ggtcatcttt gatttagaga tgataatccc atactctcaa ttgagttaaa tcagtaatct2101 gtcgcatttc atcaagatta attaaaattt gggacctgct
10、tcattcaagc ttcatatatg2161 ctttgcagag aactcataaa ggagcatata aggctaaatg taaaacacaa gactgtcatt2221 agaattgaat tattgggctt aatataaatc gtaacctatg aagtttattt tctattttag2281 ttaactatga ttccaattac tactttgtta ttgtacctaa gtaaattttc tttaggtcag2341 aagcccatta aaatagttac aagcattgaa cttctttagt attatattaa tataaaaac
11、a2401 tttttgtatg ttttattgta atcataaata ctgctgtata aggtaataaa actctgcacc2461 taatccccat aacttccagt atcattttcc aattaattat caagtctgtt ttgggaaaca2521 ctttgaggac atttatgatg cagcagatgt tgactaaagg cttggttggt agatattcag2581 gaaatgttca ctgaataaat aagtaaatac attattgaaa agcaaatctg tataaatgtg2641 aaatttttat ttg
12、tattagt aataaaacat tagtagttta5 gaattcatgtccactgcggtcctgga（5 end primer）（5端引物） 3 gtcgacttactgtattttctggaggg （3 end primer）（3端引物）,同源性检验 Homology test,http:/www.ppthi-,同源性检验 Homology test,同源序列,人类苯丙氨酸羟化酶 人类视网膜变性3（RD3），转录变异体2，mRNA 人类膜结合的转录因子的肽酶，位置2（MBTPS2），mRNA,http:/www.ppthi-,Cleavage point detection 酶
13、切位点检测,http:/www.ppthi-,寻找引物的发夹结构 searching for hairpin structures of primers,http:/www.ppthi-,寻找互补的两个引物配对 searching for complementary pairing of two primers,http:/www.ppthi-,寻找两个引物的重复序列 searching for repeated motifs of two primers,http:/www.ppthi-,分析引物的碱基和计算Tm值 analysing base distribution of a prime
14、r and calculation of Tm value,http:/www.ppthi-,二. Carrier selection and preparation 载体的选择与准备,As the size of the target gene is less than 10kb，so prepare pUC18/19 plasmid vector。 由于目的基因的大小小于10kb，所以准备pUC18/19质粒载体。,http:/www.ppthi-,The connection of the target DNA and the carrier 三目的DNA与载体连接,http:/www.
15、ppthi-,principle：use the DNA ligase to connect 5 - phosphate and 3-OH which are closed to each other 原理：用DNA连接酶把相互靠近的5-磷酸与3-OH连到一起Select sticky end connection - double digested point connection of the sticky ends，fluorescent labeling at the the plasmid cleavage sites 选择黏端连接-双酶切点的粘性末端连接，在质粒的酶切位点处进行荧光
16、标记,The recombinant DNA into a host cell for amplification - Calcium chloride import 四重组DNA转入宿主细胞进行扩增-氯化钙导入法,http:/www.ppthi-,Screening and Identification of recombinant 五重组体的筛选与鉴定,Tactics： 1）First screened cloning with a carrier 策略： 1）先筛选出带有载体的克隆 Second screening identified cloning with recombinant vector2）然后筛选鉴定出带有重组载体的克隆 Last screening identified cloning with the purpose of DNA3）最后筛选鉴定出带有目的DNA的克隆,
