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Mapua Institute of Technology Introduction to Bioelectronics and Bioinformation Engineering
School of Chemical Engineering and Chemistry DNA TECHNOLOGY AND GENOMICS
Bacterial Vectors
a. Plasmids
- circular double strands of DNAs that are
extrachromosomal. Once cell clones are identified, they are grown in
liquid culture in a large tank and then easily isolate
Cloning Eukaryotic Gene in a Bacterial Plasmid large amounts of the gene.
Ex: E. coli and human DNA
1. Isolation of vector and gene-source DNA b. Bacteriophage
- DNA containing gene of interest obtained - a virus that infects a bacterium. Recombinant
from human tissue culture and plasmid from DNA containing viral DNA and DNA of interest
R are packaged into viral particles in a test tube.
E. coli carrying two genes (amp – resistance
to ampicillin and lacZ – encoding enzyme β- Host bacterial cells are infected with
galactosidase). Plasmid has a single recombinant phage DNA, the DNA replicates
recognition sequence that lies within lacZ. within the host cells, and progeny phage are
produced when the host cell undergoes lysis.
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Mapua Institute of Technology Introduction to Bioelectronics and Bioinformation Engineering
School of Chemical Engineering and Chemistry DNA TECHNOLOGY AND GENOMICS
T-DNA
Foreign gene T-DNA with Complimentary DNA (cDNA)
inserted into inserted gene -DNA that carries the complete coding sequence for
Ti plasmid
Ti a gene but no introns.
Plasmid Recombinant Making a cDNA:
Ti plasmid Fully processes mRNA (introns removed)
extracted from eukaryotic cell nucleus.
Addition of reverse transcriptase to synthesize
Agrobacterium tumefaciens
DNA from RNA.
Agrobacterium
tumefaciens is DNA polymerase is used to synthesize a second
transformed DNA strand.
with Result is cDNA, which carries the complete
recombinant Ti coding sequence of the gene but no introns.
Recombinant
plasmid
Ti plasmid
Cloned genes are stored in DNA libraries.
Thousand of different recombinant plasmids are
Leaf discs or
produced in cloning, each carrying copies of a
protoplasts are particular segment from the initial genome. These
infected with A. sets of recombinant plasmid clones are saved in
tumefaciens such a library called Genomic libraries.
Genomic libraries:
Plasmid library
Regenerate TRANSGENIC Phage library
PLANT cDNA library
T-DNA with inserted gene is incorporated Screening library
into host genome Expression library
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Mapua Institute of Technology Introduction to Bioelectronics and Bioinformation Engineering
School of Chemical Engineering and Chemistry DNA TECHNOLOGY AND GENOMICS
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Mapua Institute of Technology Introduction to Bioelectronics and Bioinformation Engineering
School of Chemical Engineering and Chemistry DNA TECHNOLOGY AND GENOMICS
3’ GGGCCTAAGCCTTAAACTGAAGGTTATGCCCCTTAGCC 5’
5’ CCCGGATTCGGAATTTGA*
3’ GGGCCTAAGCCTTAAACTGAAGGTTATGCCCCTTAGCC 5’
5’ CCCGGATTCGGAATTTGACTTCCA*
3’ GGGCCTAAGCCTTAAACTGAAGGTTATGCCCCTTAGCC 5’
5’ CCCGGATTCGGAATTTGACTTCCAA*
Reference:
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Mapua Institute of Technology Introduction to Bioelectronics and Bioinformation Engineering
School of Chemical Engineering and Chemistry DNA TECHNOLOGY AND GENOMICS
utasevilla@mapua.edu.ph Page 6
Mapua Institute of Technology Introduction to Bioelectronics and Bioinformation Engineering
School of Chemical Engineering and Chemistry DNA TECHNOLOGY AND GENOMICS
DNA SEQUENCING
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