Gene Manipulation

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Gene Manipulation
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Gene Manipulation

• ???????????????????? genetic materials ?????????
• ?????????????? DNA ?????????????????? ??? DNA
• ????????????????????????? ????????
  Recombinant DNA
• ????????????? methodology ????????
• Recombinant DNA technology ????
• Genetic engineering ???? Gene cloning
• Clone ??????? ???????????????????????

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????????? Gene Cloning

• 1. ????? DNA ????????????????? clone ????????
  Foreign DNA ???????? DNA ????????????????????????
  ??? ???????? Vector DNA
• ?????? foreign DNA ??? ??? vector DNA ????
  enzyme ????????????
• Enzyme ?????????????? Restriction enzyme

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????????? Gene Cloning (???)

• 2. ????????? (fragment) ??? cut foreign DNA
  ???????????????? clone ??? ??????????????????
  Electrophoresis ???????????? (bands) ??? DNA
  ???????? ? ???
• ???????????????????????????????????????????? DNA
  fragment ??????????

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????????? Gene Cloning (???)

• 3. Insert DNA fragment ?????????????????? cut
  vector DNA ???? enzyme Ligase ????? DNA
  ?????????????? ???????? Chimeric DNA ????
  Recombinant DNA
• 4. Introduce Recombinant DNA ???????? host cells
  ??? cloning vector ??????? transformation ????
  ???????? ?
• host cells ???????????? E. coli ??? yeast

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Recombinant DNA
First cloning
Principle of cloning
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????????? Gene Cloning (???)

• 5. Recombinant DNA ????? host ???????????????????
  ???????????????????????????????????????????host
  ???????? identical copies ??? recombinant DNA
  ???????? ???????? Amplification
• 6. ???????????????? (screening) host cells ??
  recombinant DNA

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Cloning Vectors

• 1. Plasmid
• 2. Bacteriophage
• 3. Cosmid
• 4. Phagemid
• 5. Artificial chromosome

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1. Plasmid vectors

• Plamids ???? extrachromosome ?? microorganisms
  ???????? bacteria
• ???? double-stranded circular ???????
• ???? 1 kb (1 kb 1000 bp) ??? 200 kb
• ?? antibiotic-resistance genes ????????????????
  markers ?????????????? cloning ??? positive
• pBR322 ???? plasmid ?????????????? cloning

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1.1 pBR322

• ???? 43. Kb
• ?? Ampicillin resisteant gene ??? Tetracyclin
  resistant gene
• ?? restriction sites ?????????????????????
  cloning ???? EcoRI, PstI, BamHI
• ?? Origin of replication
• ?????? bacteria ??? Transformation

pBR322
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Cloning in pBR322
Amplification
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1.2 pUC8

• Plasmid ??? carry 2 markers
• 1. lacZ gene code ??? b-galactosidase ???
• 2. Amplicillin resistant gene
• culture E.coli host ?? X-galactopyranoside
• (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside)
  

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1.3 Yeast episomal plasmids / YEps

• 2 um plasmid
• ???? 6 kb
• ?? gene resist
• ??? inhibitors
• ???? copper ??? methotrexate
• ??? leu2 gene ???? marker

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Cloning in yeast
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1.4 Ti plasmid

• Ti plasmid ???? T-DNA ?? Agrobacterium
  tumefaciens
• ?????
• integrate ????
• plant chromosomal
• DNA
• produce callus
• ???? cancer growth ???? crown gall ?????

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Plant clonimg using Ti plasmid
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2. Bacteriophage vectors

• Double-stranded virus ??????????????????? Linear
  duplex ???
• ?????????? Bacteriophage Lambda(l) ???? 50 kb
• ?? Linear duplex form ???????????????????? 2
  ??????????????????? Single strand ???? 12
  nucleotides ???????? Cohesive ends ???? COS sites
  
• Cos sites ?? complementary ?????? ??????
  circular duplex DNA ???? pack ???? capsule
  ???????? bacteriophage

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2.1 Charon phage vector

• l ?? genes 50 ???
• ????????? growth
• l package DNA ???
• ???? circular form ???
• 78-105
• l vector ??? insert ??? 12-25 ?????????????
  clone eukaryote ???? ?? Genomic library

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2.2 M13 vector

• single-stranded phage
• Rolling circle replication
• ??? insert ???????
• single-stranded version ??? cloned gene ???????
  300-1000 bases
• ??? cloning ????? sequence DNA

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3. Cosmid vector

• Vector ??? construct ??????????
• 1. Plasmid drug-resistant marker, origin of
  replication ??? restriction sites ?????
  replication ????????? plasmid
• 2. l phage cos sites ?????? packaging cloned
  DNA ??????? phage chromosome ?? in vtro

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Cosmid vector

• cosmid ?????? 4-6 kb
• cosmid ????????? insert foreign DNA ?????????? 35
  - 50 kb ?????????? clone gene ???????????
  eulararyote

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4. Pagemid vectors

• Vector ??? construct ????? characteristics ?????
  cosmid ??? ????????? phage ??? plasmid
• ?? multiple cloning site insert ??????? lacZ gene
• ?? origin of replication ??? single-stranded f1
  ????? M13 ????? package ??? single-stranded
  phagemid DNA

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Transcription vector / Bluescript vector / pBS

• ?? Multiple cloning site
• ?? 2 RNA polymerase
• promoters ???
• T3 promoter ?????????
• ??? T7 promoter
• ????????????
• ?????? transcribe RNA ?? in vitro
• ???????? translation in vitro

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5. Artificial chromosome

• ???? Construct ??????? cloning ?? yeast ????????
  Yeast Artificial chromosome / YAC
• ???? mini-chromosome ??????????
• 1 centromere, 2 telomeres, origin of replication,
  ??? seletable marker gene(s)
• ???????? chromosome segregation ??????? meiosis
• ??? cloning DNA ?????????????? 150 kb

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YAC vector

• Yeast artificial chromosome vector

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DNA manipulative enzymes

• Enzymes ????????? DNA ????? specific ???????
• ????????? ?? 2 ????????
• 1. Restriction endonucleases
• 2. DNA ligases

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Restriction Endonuclease

• Type II restriction endonuclease
• recognize specific nucleotide sequences
• cut 4-8 nucleotide sequence
• cut ends 2 types
• Blunt ends ???? AluI
• Stricky ends ???? EcoRI

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Cut ends by restriction endonucleases
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DNA ligase

• ?????? DNA ??????????????
• Sticky ends efficiency ??????????
• Blunt ends efficiency ????????

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DNA library

• ??? Set ??? DNA clones ???????????????????????????
  ???????????? genes
• Genomic DNA library ??? 1 genome ??????? genes
  ?????????? ????????????? ?
• Chromosome-specific DNA library ??? 1
  chromosome
• Complementary DNA library ??? cDNA

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Application ??? Gene manipulation

• 1. In vitro Site-specific mutagenesis
  ???????????????? genes
• 2. Physical maps / restriction maps of DNA
  molecule
• 3. Nucleotide sequences of gene ??????? base
  ??? gene
• 4. Identification of genes genetic diseases
• 5. Human gene therapy ????????????????????

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• 6. Paternity tests DNA fingerprints, forensic
  application
• 7. Production of proteins hormones, enzymes
• 8. Transgenic plants crown gall, herbicide ???
  insecticide resistant plants
• 9. Transgenic animals protein production for
  human ???????????????????????????

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• ???????? ?????????????? Gene cloning

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DNA Sequencing
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Cloning in Plants
Glyphosate-telerant petunia
Luciferaseplant ???????????
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DNA Fingerprintings Identify ????? / ???-???
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Gene Therapy ????????????????????
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Gene Transfer ???? Identify gene ??????????????
????????? gene cloning
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Transgenic mouse
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Production ??? Somatostatin (action ????? growth
hormone) ?? recombinant E.coli