Recombinant
DNA
Technology
,Contents
1 Introduction ............................................................................................................................. 1
1.1 Genetic Engineering ......................................................................................................... 1
1.2 Applications ..................................................................................................................... 1
2 History ..................................................................................................................................... 2
3 Developing a Recombinant DNA ............................................................................................ 3
3.1 Four Steps of Recombinant DNA Technology ................................................................ 4
3.1.1 DNA Fragmentation.................................................................................................. 4
3.1.2 Ligation of Fragment of Interest with the Vector ..................................................... 4
3.1.3 Introduction into Host ............................................................................................... 5
3.1.4 Selection and Screening ............................................................................................ 5
4 DNA Manipulating Enzymes .................................................................................................. 6
4.1 DNA Polymerases ............................................................................................................ 6
4.2 Ligases .............................................................................................................................. 8
4.3 Nucleases .......................................................................................................................... 9
4.3.1 Fragmentation of Linear DNA ................................................................................ 11
4.3.2 Cutting a Circular DNA .......................................................................................... 12
4.4 End Modifying Enzymes ................................................................................................ 13
4.4.1 Alkaline Phosphatase .............................................................................................. 13
4.4.2 Polynucleotide Kinase ............................................................................................ 14
4.4.3 Terminal Deoxynucleotidyl Transferase................................................................. 14
5 DNA Cloning and Cloning Vectors ...................................................................................... 15
5.1 Vectors Based on E. coli Plasmids ................................................................................. 16
5.1.1 pBR322 Vector ....................................................................................................... 16
i
, 5.1.2 pUC8 Vector ........................................................................................................... 18
5.2 Bacteriophage Lambda Based Vector ............................................................................ 20
6 Verification of Recombinant Clones .................................................................................... 23
6.1 Verification of Recombinant Clones Using PCR........................................................... 23
6.2 Verification of Recombinant Clones Using Restriction Endonucleases ........................ 24
7 Genomic Libraries and Library Screening ............................................................................ 25
7.1 Genomic Library ............................................................................................................ 25
7.2 cDNA Library ................................................................................................................ 25
7.2.1 Constructing cDNA ................................................................................................ 26
7.3 Library Screening ........................................................................................................... 26
7.3.1 Library Screening Using Homologous Probes ....................................................... 27
7.3.2 Library Screening Using Heterologous Probes....................................................... 27
7.3.3 Library Screening Using Degenerate Probes .......................................................... 28
7.3.4 Immunological Screening ....................................................................................... 28
7.3.5 Screening Libraries Based on Gene Function ......................................................... 29
8 Cloning a PCR Product ......................................................................................................... 30
8.1 Cloning of PCR Product Using Adapter Primers ........................................................... 30
8.2 TA Cloning..................................................................................................................... 32
8.3 Gateway Cloning ............................................................................................................ 32
9 Plant Transformation ............................................................................................................ 39
9.1 Agrobacterium tumefaciens............................................................................................ 39
9.2 Ti-Plasmid and its Components ..................................................................................... 39
9.3 Natural Infection Process of A. tumefaciens .................................................................. 40
9.4 Challenges Associated with Natural Ti-Plasmid and their Solutions ............................ 42
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, 9.5 Developing a Transgenic Plant ...................................................................................... 43
9.5.1 Tissue Culture Method ............................................................................................ 43
9.5.2 Floral Dip method ................................................................................................... 44
9.6 Confirmation of Successful Transformation .................................................................. 44
9.6.1 Checking Integration of Gene of Interest ................................................................ 44
9.6.2 Checking the Expression of Gene of Interest ......................................................... 45
9.6.3 Protein Detection .................................................................................................... 45
9.6.4 Confirmation of Traits ............................................................................................ 45
9.7 Markers for Transgenic Plants ....................................................................................... 45
10 Transgenic Animals .............................................................................................................. 45
10.1 Methods of Animal Tranformation ................................................................................ 46
10.1.1 DNA microinjection method................................................................................... 46
10.1.2 Embryonic Stem Cell Method ................................................................................ 47
10.2 Ensuring Targeted Integration ........................................................................................ 47
10.3 Developing Knockout Mice ........................................................................................... 49
10.3.1 Process of developing a KO mouse ........................................................................ 49
iii
DNA
Technology
,Contents
1 Introduction ............................................................................................................................. 1
1.1 Genetic Engineering ......................................................................................................... 1
1.2 Applications ..................................................................................................................... 1
2 History ..................................................................................................................................... 2
3 Developing a Recombinant DNA ............................................................................................ 3
3.1 Four Steps of Recombinant DNA Technology ................................................................ 4
3.1.1 DNA Fragmentation.................................................................................................. 4
3.1.2 Ligation of Fragment of Interest with the Vector ..................................................... 4
3.1.3 Introduction into Host ............................................................................................... 5
3.1.4 Selection and Screening ............................................................................................ 5
4 DNA Manipulating Enzymes .................................................................................................. 6
4.1 DNA Polymerases ............................................................................................................ 6
4.2 Ligases .............................................................................................................................. 8
4.3 Nucleases .......................................................................................................................... 9
4.3.1 Fragmentation of Linear DNA ................................................................................ 11
4.3.2 Cutting a Circular DNA .......................................................................................... 12
4.4 End Modifying Enzymes ................................................................................................ 13
4.4.1 Alkaline Phosphatase .............................................................................................. 13
4.4.2 Polynucleotide Kinase ............................................................................................ 14
4.4.3 Terminal Deoxynucleotidyl Transferase................................................................. 14
5 DNA Cloning and Cloning Vectors ...................................................................................... 15
5.1 Vectors Based on E. coli Plasmids ................................................................................. 16
5.1.1 pBR322 Vector ....................................................................................................... 16
i
, 5.1.2 pUC8 Vector ........................................................................................................... 18
5.2 Bacteriophage Lambda Based Vector ............................................................................ 20
6 Verification of Recombinant Clones .................................................................................... 23
6.1 Verification of Recombinant Clones Using PCR........................................................... 23
6.2 Verification of Recombinant Clones Using Restriction Endonucleases ........................ 24
7 Genomic Libraries and Library Screening ............................................................................ 25
7.1 Genomic Library ............................................................................................................ 25
7.2 cDNA Library ................................................................................................................ 25
7.2.1 Constructing cDNA ................................................................................................ 26
7.3 Library Screening ........................................................................................................... 26
7.3.1 Library Screening Using Homologous Probes ....................................................... 27
7.3.2 Library Screening Using Heterologous Probes....................................................... 27
7.3.3 Library Screening Using Degenerate Probes .......................................................... 28
7.3.4 Immunological Screening ....................................................................................... 28
7.3.5 Screening Libraries Based on Gene Function ......................................................... 29
8 Cloning a PCR Product ......................................................................................................... 30
8.1 Cloning of PCR Product Using Adapter Primers ........................................................... 30
8.2 TA Cloning..................................................................................................................... 32
8.3 Gateway Cloning ............................................................................................................ 32
9 Plant Transformation ............................................................................................................ 39
9.1 Agrobacterium tumefaciens............................................................................................ 39
9.2 Ti-Plasmid and its Components ..................................................................................... 39
9.3 Natural Infection Process of A. tumefaciens .................................................................. 40
9.4 Challenges Associated with Natural Ti-Plasmid and their Solutions ............................ 42
ii
, 9.5 Developing a Transgenic Plant ...................................................................................... 43
9.5.1 Tissue Culture Method ............................................................................................ 43
9.5.2 Floral Dip method ................................................................................................... 44
9.6 Confirmation of Successful Transformation .................................................................. 44
9.6.1 Checking Integration of Gene of Interest ................................................................ 44
9.6.2 Checking the Expression of Gene of Interest ......................................................... 45
9.6.3 Protein Detection .................................................................................................... 45
9.6.4 Confirmation of Traits ............................................................................................ 45
9.7 Markers for Transgenic Plants ....................................................................................... 45
10 Transgenic Animals .............................................................................................................. 45
10.1 Methods of Animal Tranformation ................................................................................ 46
10.1.1 DNA microinjection method................................................................................... 46
10.1.2 Embryonic Stem Cell Method ................................................................................ 47
10.2 Ensuring Targeted Integration ........................................................................................ 47
10.3 Developing Knockout Mice ........................................................................................... 49
10.3.1 Process of developing a KO mouse ........................................................................ 49
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