基因選殖的策略PPT課件[通用]

1、基因選殖的策略原核生物-細菌萃取chromosome DNAPartial digestion DNA(選一種限制酶)Vector (去磷酸根)(降低self-ligation)Ligation, trnasformation藍白篩選活性篩選真核生物?M - Marker : DNA/HindIII1 - Streptomyces PMS502 chromosome 2 - 1/BamHI (mg DNA/0.18 unit)3 - 1/BamHI (mg DNA/ 0.06 unit)4 - 1/BamHI (mg DNA/ 0.02 unit)5 - 1/BamHI (mg DNA/ 0.

2、007 unit)23.19.46.64.42.32.0M 1 2 3 4 5ATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCLigationTransformationPicking white colonies選殖基因的定序Clone確認定序primer的選擇Vector上的forward reverse序列Ex: pUC18第二次定序自行設(shè)計primer序列的組合(正股 反股)Determining DNA sequences1975 Frederick Sanger dideoxynucleoside triphosp

3、hates (ddNTPs)定序完成後的工作上網(wǎng)比對序列blast確認所clone的基因預(yù)測ORF (open reading frame) DNA-STAR VECTOR NTI 9.0ORF的確認Ribosome binding site (RBS -6 -12 GA rich)蛋白的分子量與基因長度相關(guān)性TATA box (-35 -10)設(shè)計primers subclone, expressionExpression vector 的選擇(pET, pQE, pGEX4T)基因產(chǎn)物的純化設(shè)計Primers PCR基因產(chǎn)物前的分析工作解讀限制酶圖譜Primer序列加上限制酶切位選擇哪種限

4、制酶切位Multi-cloning site有的切位轉(zhuǎn)錄或轉(zhuǎn)譯起始位置的考量(與載體有關(guān))primer設(shè)計時要注意序列 in frame實做練習(xí)ATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGGAGCCGGGGAGCACATTGGCGGCACCGTGAGAGCGCTCTCAAAGGTCCCGCGTCCCCC 660GCTCCCCGCTCCCGAGAGGCCACCCATGAGTGATTCCTCCGGCACCCCCAGACCCCGTTC 720 M S D S S G T P R P R S CCACTCCCGCCCCCGCT

5、CCCGCTCCGTGCGCCGCGCCCTCATGGCGGCCGTCGCGACGTT 780 H S R P R S R S V R R A L M A A V A T FCGGCCTCGCCGCGGCCGTCGCCACCGCGGCCACCGGCCCCGCGGACGCCTCCGCCCCGGC 840 G L A A A V A T A A T G P A D A S A P AGCCGCCCTCCGGCTGGTCGCAGGTGTTCCTCGACGACTTCGACGGCGCGGCCGGAAGCAG 900 P P S G W S Q V F L D D F D G A A G S SCGTCA

6、ACACCGCCAACTGGCAGTTCGACACCGGCACTTCGTACCCCGGCGGGGCAGGCAA 960 V N T A N W Q F D T G T S Y P G G A G NCTGGGGCACTGGCGAGGTCGAGTCGATGACGTCGAGCACCAGCAATGTCTCCCTCGACGG 1020 W G T G E V E S M T S S T S N V S L D GCAACGGCGATCTGCTCATCACCCCGCGCCGCGACGCGTCCGGCAACTGGACCTCCGGCCG 1080 N G D L L I T P R R D A S G N

7、W T S G RGATCGAGACCACCCGCACCGACTTCCAGCCCCCGGCCGGCGGCAAGCTGCGCGTCGAGGC 1140 I E T T R T D F Q P P A G G K L R V E AGCGCCTGCAGATGCCGAACGTGACCGGGGACGCGGCCGCCGGGTACTGGCCCGCGTTCTG 1200 R L Q M P N V T G D A A A G Y W P A F WGATGCTGGGCGCGCCCTTCCGCGGCAACTACCAGAACTGGCCCGGCGTCGGCGAGCTGGA 1260 M L G A P F R G

8、 N Y Q N W P G V G E L DCATCATGGAGAACGTCCAGGGCCTGAACAAGACCTGGGCCACCATGCACTGCGGGACCAG 1320 I M E N V Q G L N K T W A T M H C G T SCCCCGGCGGGCCCTGCAACGAGACCAGCGGCATCGGCAATTTGACCGCCTGCCCGAACAC 1380 P G G P C N E T S G I G N L T A C P N TCACCTGCCATTCGGGCTTGCACACCTACACCATGGAGTGGGACCGCTCGGTCAGCCCCGA 1440

9、T C H S G L H T Y T M E W D R S V S P EGGCGATCCGCTTCTCCGTCGACGGCGTCACCTACCAGACGGTCACGGCGAATCATATGGA 1500 A I R F S V D G V T Y Q T V T A N H M DCGCGGTCACCTGGACCAACGCCACGAACCACGGTTTCTTCGTTATCCTGAACGTGGCGAT 1560 A V T W T N A T N H G F F V I L N V A MGGGCGGCGGCTTCCCGGGTGCCTTCGGCGGCGGCCCGACCGGCGCCACCGA

10、GCCCGGCCA 1620 G G G F P G A F G G G P T G A T E P G HCCCCATGGTCGTCGACTACGTCCAGGTGCTCCAGTCCTCGGGCGGCGGGGGCGGCGGGGG 1680 P M V V D Y V Q V L Q S S G G G G G G GCGGGGGCACCACTCCCCCGCCGACCGGCGACCGGGACGCGTACGGTCAGATCCAGGCGGA 1740 G G T T P P P T G D R D A Y G Q I Q A EGTCGTACGACGGCCAGTCCGGTGTCGCCACCGAGAC

11、CACCACGGACACCGGCGGCGGACA 1800 S Y D G Q S G V A T E T T T D T G G G QGGACATGGGCTACCTCGCCAATGGCGACTGGGCGCTGTACAAGGGCGTGAACTTCGGCTC 1860 D M G Y L A N G D W A L Y K G V N F G SGACACCGGCCACCCAGTTCTACGGTCGGGTGGCGAGCGGCGCGGGCGGCGGGGTCAGCGG 1920 T P A T Q F Y G R V A S G A G G G V S GCCTGGTGGAGGTGCGGCTGGACAGCCGTACGAACGCGCCGATCGGGAGCTTCGCGGTGGG 1980 L V E V R L D S R T N A P I G S F A V GCGACACCGGGGGCTGGCAGTCCTGGCGGACGGTTCCGGCCAACATCGGGTCCGTCACCGG 2040 D T G G W Q S W R T V P A N I G S V T GC