Gene Technology

Gene Technology
 Procedures involving the manipulation of DNA
Includes
taking genes from one organism and placing
them into another
genetic fingerprinting
Also called genetic engineering or recombinant
DNA technology
 Stages/Techniques
 Many techniques involved in gene technology
can be demonstrated using the example of human
insulin production by genetically modified
bacteria.
 Required tasks:-
 Isolate the human insulin gene.
 Insert the gene into a suitable host bacterium
using as vector.
 Produce many copies of the bacterium which will
produce insulin.
 Separate and purify the insulin (downstream
processing)
 Stage 1 Method A
Cutting out the gene
 This method involves cutting the gene out from a
complete chromosome.
 Electrophoresis and DNA probes are used to
identify the gene in donor DNA.
 The gene is cut using restriction endonucleases.
 This method has limitations due to different
regulatory genes in prokaryotes and eukaryotes
and the presence of introns.
 Restriction Endonucleases
 Molecular scissors
 Found naturally in bacteria
 Use to destroy viral DNA
 Over 400 types now been obtained
 Recognise a specific base sequence
 Cut DNA unevenly to produce “sticky ends” or
make straight cuts to form blunt ends
EcoR1 – from E. coli
 Stage 1 Method B
Make a copy of the gene
 mRNA is obtained from cells producing insulin
 Using the enzyme reverse transcriptase
 A DNA sequence is copied from mRNA
 Making single stranded copy DNA (cDNA)
 DNA polymerase uses free nucleotides to make
the complementary strand
 This method is preferable as the cell has many
mRNA copies versus one gene in DNA and the
mRNA strand is the same length as the gene and
doesn’t have to be cut
Reverse transcriptase – found in
retroviruses (HIV)
 Stage 2 A suitable vector
 Vectors are molecules of DNA which are used to
carry the foreign gene into the host DNA
 Examples include bacterial plasmids and phage
viruses
 Plasmids
 Plasmids are circular pieces of double stranded
DNA found in bacteria in addition to the main
DNA.
 May contain useful gene such as antibiotic
resistance.
 Can enter bacteria and plasmid DNA can
replicate when bacteria undergo binary fission
Insertion of gene into vector
 DNA ligase
 Both the DNA (if using method A) and the
plasmid are cut using the same restriction
endonuclease
 The “sticky ends “ of the plasmid and the gene
are complementary and are attracted to each
other
 Hydrogen bonds form between complementary
bases
 DNA ligase is used to glue the cut ends
 Stage 3 – using the vector to insert
the gene into the host
 Plasmids are removed from the bacterial host
cells.
 Recombinant plasmids are incubated with the
host bacteria
 Calcium ions, temperature and electrical shocks
can be used to make the bacterial cell walls more
permeable and take up the plasmid
 Some bacteria take up the recombinant plasmids
to become genetically modified
 Genetic Screening
 Only some of the bacteria have been modified and
need to be separated out
 DNA probes or genetic markers can be used
 The human insulin gene in inserted into the middle
of a gene for antibiotic resistance
 Modified bacteria are not antibiotic resistant and
can be identified by replica plating
Replica Plating
 Stage 4 Multiplication of the Host
Cell
 Modified bacteria are grown in industrial
fermenters on a large scale
 Bacteria are allowed to reproduce asexually and
produce clones
 Plasmids also replicate each time the cell divides
 The bacterial cells express the human insulin
gene
Stage 5 Downstream Processing

 Insulin made by the bacteria is excreted into the

culture medium
 The insulin then needs to be extracted and
purified
 Human Insulin
 Over 2 million people inject insulin daily
 Previously used cow or pig insulin from dead
animals
 Different chemically
 Immune response destroyed this insulin
 Fermentation produces large quantities cheaply
 No ethical/religious/moral issues