Recombinant DNA

Technology

Kashif Iqbal Sahibzada

Techniques used in rDNA technology

• Gel electrophoresis
• Cloning libraries
• Restriction enzyme mapping
• PCR
• Nucleic Acid Hybridization
• DNA Microarrays
Cloning plasmid
Polymerase chain reaction
 Inserting DNA into cells
• 1. Transformation
• 2. Electroporation
– Electric current make pores in the cell so DNA
can enter
• 3. Protoplast fusion
– Cells with no cell wall can be fused and
natural recombination may occur
• 4. Microinjection
Protoplast fusion
 How are genes isolated?
• 1. Gene libraries
– Digestion of entire genome with restriction
enzymes
– Insert fragments into vectors and put the
vectors into bacterial cells
• 2. Complementary DNA (cDNA)
– Eukaryotic gene derived from mRNA made
with reverse transcriptase
– Lacks introns only exons
Gene Library
Complementary DNA
 Selecting the clone
• Need to next be able to find the cell with the
gene of interest
• Selecting the clone of interest is often done with
marker genes
• Genes are spliced into plasmids carrying genes for
ampicillin resistance and β-galactosidase
• Colonies that grow with special characteristics are
selected as potential clones with the gene you
want!
Selecting recombinant bacteria
 Selecting the clone
• After candidate colonies are identified the
one with the gene of interest must be
selected
• DNA probes
• Pieces of single stranded DNA
complementary to the desired gene are
made and labeled with a radioactive
element
• Helps identify the target gene
DNA Probes
 Making a gene product
• Put the gene into a bacteria like E. coli and get the
product made
– Toxic by-products from Gram- cell wall
– Usually no secretion of product by Gram - cells
• Yeast cells
– Better secretion of product
• Mammalian cells
– Good source for protein products
– Little risk of toxins and allergy
Applications of rDNA technology

• Agriculture: growing crops of your choice (GM food),

pesticide resistant crops, fruits with attractive colors, all
being grown in artificial conditions
• Pharmacology: artificial insulin production, drug delivery to
target sites
• Medicine: gene therapy, antiviral therapy, vaccination,
synthesizing clotting factors
• Other uses:fluorescent fishes, glowing plants etc
Products of genetic engineering
Products of genetic engineering