1 I Recombinant DNA Gene cloning F II
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I. ������� Recombinant DNA (Gene cloning( F II. �������� gene cloning F III. ������� Gene cloning F 3
���� Recombinant DNA 5
II. �������� Gene Cloning . 1 Gene ������� foreign DNA 2. DNA ������� Vector DNA 3. Restriction endonuclease 4. DNA ligase ����� 2 nucleotides 5. Host cells ������ recombinant DNA �������� 6
DNA gel electrophoresis ������� ��� DNA 9
. 3 Insert DNA fragment ��������� cut vector DNA ���� enzyme Ligase ����� DNA ������� Chimeric DNA ���� Recombinant DNA. 4��Recombinant DNA ���� host cells ��� cloning vector ������� Transformation �������� � host cells ������ E. coli ��� yeast 5. Recombinant DNA ����� host ��������� Amplification ��������� 10 �������� host
6. �������� (screening) host cells �� recombinant DNA 12
Cloning Vectors. 1 Plasmid. 2 Bacteriophage. 3 Cosmid. 4 Phagemid 5. Artificial chromosome 13
. 1 Plasmid vectors Plasmids F ���� extrachromosome �� microorganisms ���� bacteria F ���� double-stranded circular ������� F ���� ~ 1 kb (1 kb = 1000 bp) ��� 200 kb F �� antibiotic-resistant genes �������� markers ������� clones ��� positive 14
1. 1 Plasmid p. BR 322 ���� 4. 3 Kb F �� Ampicillin resisteant gene Tetracyclin resistant gene F �� restriction sites ����������� cloning ���� Eco. RI, Pst. I, Bam. HI F �� Origin of replication F ���� cloning vector �� prokaryote F ������ bacteria ��� Transformation F 15
F Bacterial cloning ������ plasmid p. BR 322 16
1. 2 Yeast episomal plasmids / YEps ����� 2 um plasmid (����� ) ��� yeast F ���� 6 kb F �� resistant gene ��� inhibitors ���� copper ��� methotrexate F marker ��� leu 2 gene F ������� cloning vector �� eukaryote F Transform yeast 17 F
F Yeast cloning ����� � plasmid ���� �LEU 2 gene ���� marker 18
1. 3 Ti plasmid ���� T-DNA �� Agrobacterium tumefaciens �� ��� F integrate ���� plant chromosomal DNA F ��������� (callus) ���� cancer growth ���� crown gall ���� F 19
Gene cloning ������ Ti plasmid F ���� T -DNA (tumor DNA) �� bacteria infect (���� ) ���� chromosome ������ --> crown gall --> ���� F 20
. 2 Bacteriophage vectors Double-stranded virus ��������� �Linear duplex F ����� Bacteriophage Lambda (l ( ���� ~ 50 kb F l �� genes ~ 50 % ���� 2 ������� linear duplex ������ growth F ������ ��� ����� growth 21 F
�� Linear duplex ��������� 2 ��������� Single strand ���� 12 nucleotides ���� Cohesive ends ���� COS sites F Cos sites ��complementary ������ circular duplex DNA ���� pack ��� capsule ���� bacteriophage ���� F F l vector ��� insert ��� 12 -25 % �������� cloning 23
2. 2 M 13 vector Single-stranded phage F ��� insert ������� single-stranded DNA ������� 300 -1000 bases F Rolling circle replication ��� double strand F ������ bacteria ����� single strand F ������� cloning ����� sequence DNA 24 F
. 3 Cosmid vector Vector ���� (construct) ���� 1. Plasmid : drug-resistant marker, origin of replication ��� restriction sites ����� replication ����� plasmid 2. Phage : cos sites ������ packaging cloned DNA ������� phage chromosome �� in vtro 25
cosmid ������ 4 -6 kb F construct ����� polycloning site F cosmid ����� insert foreign DNA ����� 35 50 kb F ����� clone gene F 26
. 4 Pagemid vector Vector ��� construct ���������� cosmid ��������� phage ��� plasmid F �� multiple cloning site insert ������� lac. Z gene F �� origin of replication ��� single-stranded f 1 ����� M 13 ����� package ��� single. F 27
Bluescript vector / p. BS ������� Transcription vector F �� Multiple cloning site F �� 2 promoters ��� : T 3 promoter ����� ��� : T 7 promoter ����� F ������ transcribe RNA �� in vitro F ����� translation in vitro F 28
. 5 Artificial chromosome ���� Construct ����� ��cloning �� yeast ���� Yeast Artificial chromosome / YAC F ���� mini-chromosome ����� F 1 centromere, 2 telomeres, origin of replication, ��� seletable marker gene(s) F F ���� chromosome segregation ������� meiosis ��� cloning DNA ������� 150 kb 29
Yeast Artificial chromosome / YAC 30
Restriction Endonuclease recognize specific nucleotide sequences F ����� 4 -8 nucleotide �������� F 31
F Resstriction enzymes ��� DNA ������ � 2 ��� Blunt end : Sticky end : 32
DNA ligase ������� 2 nucleotide ��� 2 DNA ������� F Sticky ends : efficiency ����� F Blunt ends : efficiency F 33
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Application ��� Gene Recombinant Technology . 1 Physical maps / restriction maps of DNA molecule �������� gene �� chromosomes 2. Nucleotide sequences of gene ������� base ��� gene 3. In vitro site-specific mutagenesis ��������������� genes ���� 37
. 6 Production of proteins ���� proteins, hormones ��� enzymes ��� bacteria ���� host ��� recombinant DNA ����������������� 7. Transgenic organisms (plants & animals) ���� GMO (Gene modified organism) crown gall, herbicide ��� insecticide resistant plants ���������� ����� (����� ) ��� 39
������ Gene Recombinant Technology 40
Genetic Maps 41
DNA Sequencing 42
Cloning Growth Hormone Gene 43
Gene Therapy : ������� 44
Transgenic Animal ������ DNA ���� microinjection ������ Transgenic mouse (���� ) 45
Transgenic Plants Glyphosate-tolerant petunia Luciferase plant 46 ����
DNA Fingerprintings : Identify ����� / ��� ������ DNA Fingerprintings 47
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