UPCSE Biology

Lecture Outline :
Recombinant DNA Technology

Introduction
Genetic code is universal. Transfer of genes from donor to host via a vector. Production of a
modified organism involves the location, isolation, modification and transfer of the required
gene.

Tools of Genetic Engineering

Separation of DNA fragments by gel electrophoresis. Restriction enzymes cut DNAat
specific sites producing ‘sticky’ or ‘blunt’ ends. Restriction enzymes form the bacterial
immune system. Use of restriction enzymes as molecular scissors. DNA ligase joins together
DNA fragments. Role of ligase in DNA replication and repair.

Location and Identification of Genes

Digesting and separation of DNA. Use of DNA and RNA fragments as probes. Southern and
Northern blotting. Use of radioactivity and/or alkaline phosphatase markers.

Isolation of Genes
Synthesis of DNA oligonucleotides. Use of restriction enzymes to isolate fragments.
Polymerase chain reaction (PCR). Production of cDNA from RNA by using reverse
transcriptase. cDNA contains only expressed sequence. Reverse transcriptase as a retro virus
enzyme such as HIV.

Modification and transfer of DNA

Plasmids as vectors. Sticky ends ligase to splice DNA into a vector. Transfer of plasmid to a
new host. Use of viruses as vectors. Physical and chemical transfer methods. Use of antibiotic
resistance genes to select transformed bacteria. Use of promoter sequences to give expression
of the transferred gene.

Applications
Production of human insulin in bacteria. Milk as a source of proteins that cannot be made in
bacteria. Examples alpha-1-antitrypsin and factor VIII production in milk. Humanised
monoclonal antibodies as anticancer agents.

General Support
Covered in section 18.9, 18.10, 18.11 of your text book.
Monoclonal antibodies 15.6 of your text book
Antibiotic resistance 15.9 of your text book
HIV 15.2 and 17.1 of your text book
There is no need for detailed knowledge of the experiment protocol of southern blotting nor
the genetic basis of DNA fingerprinting and the cloning of mammals. Details of antibiotic
action and HIV life cycle beyond reverse transcription are not needed in detail nor is detail on
monoclonal antibody production.