Transcript Genetic Techniques 1

Title: Genetic Techniques 1
Learning question: What are the applications of genetic engineering?
Starter: Can you think of any real life applications of genetic engineering?
Learning Objectives
(a) outline the function of restriction enzymes
(restriction endonucleases) and ligase enzymes in
separating and joining specific DNA sequences;
(b) describe, with the aid of diagrams, the
palindromic nature of restriction enzyme
recognition sequences;
The role of restriction and ligase
enzymes
• Genetic engineering – replace,
modify or add DNA coding
sequences to a living organism.
• Uses enzymes to “cut” up parts of
DNA from one organism and
“stick” them into another.
• Resulting new organism will
incorporate genes and therefore
proteins from imported DNA.
The role of restriction and ligase
enzymes
3 main stages to GE:
1. Isolating and identifying gene>
protein
2. Putting isolated gene into
another organism using a vector
3. Cloning the organism to make
several copies of the imported
gene product.
The role of restriction and ligase
enzymes
• Restriction enzymes (restriction
endonucleases) cut DNA at particular
sequences of bases.
• Two types:
– Enzymes that cut straight across – blunt ends
– Enzymes that cut in a staggered way – sticky ends
(more useful)
Sticky ends
• Short stretches of single stranded DNA are complementary to each other.
• If both ends are cut with the same enzyme, the sticky ends will stick
together by complementary base paring, forming hydrogen bonds
• Annealing is the name given to the process by which sticky ends form
hydrogen bonds.
Restriction fragments
• DNA has several recognition sites where restriction
enzymes can “cut” at very specific points.
• These restriction sites are only 4-8 base pairs long, so
they are specific sites.
• Cuts at restriction sites produces lots of DNA fragments
of different lengths.
• The resulting fragments of DNA are known as
restriction fragments.
Bacteria and DNA
• Restriction enzymes are produced naturally by
bacteria as a defence mechanism against
viruses.
• The restrict viral growth
• Restriction enzymes used in GE are named
after the bacteria it came from, e.g. EcoR1 –
e.coli strain R was the first to be identified.
Cutting and pasting
• Once DNA fragments have been
annealed to DNA of another
organism, the broken DNA needs to
be repaired.
• Remember annealing is the
formation of complementary base
pairs – this is done by weak
hydrogen bonds.
• DNA ligase enzymes repair broken
DNA by forming covalent bonds
between the new sections.
Palindromic recognition sites
• Words or phrases that can be read the same in
both directions are described as palindromic
• Level, Hannah etc
• Restriction enzymes cut DNA at palindromic
sequences e.g. GGCCCCGG
restriction endonuclease enzymes
ligase
enzymes
restriction endonuclease enzymes
Describe the stages of
genetic engineering in
this diagram
Recombinant DNA Technology
The desired gene is
isolated in human
_________
Desired Gene cut out of
Chromosome using
__________
_________
restriction
endonuclease
Gene sealed in
ligase
Plasmid using ____.
Which joins ______
plasmid
ends together.
Bacterial cell with a
plasmid selected
__________
Plasmid removed and cut
restriction
Open using ________
__________.
endonuclease
Recombinant DNA Technology
Plasmid inserted into
host
bacterium
________
_______
cell
_______
Recombinant
bacteria
Allowed to
multiply
________
rapidly
DNA _______
probe
______
Used to check the
gene is present
Bacteria ________
identified
expresses the gene
that _______
as a protein which
is then propagated
_________.
Human proteins produced by this method include _____ (trade name humulin) ,
_____ ______ ______ (HGH)
Marker genes
• Only a small percentage of cells take up the recombinant
DNA.
• Identifying these cells is important. This can be done with
marker genes.
• Marker genes, when expressed, display easily observable
characteristics.
• When recombinant DNA is transferred into a vector of the
host cell, the marker gene goes with it. This means that if
we can see the expression of MG we know that GE has
been succesful.
Antibiotic resistance markers and
reporter systems
• Marker genes can code for resistance to
particular antibiotics.
• GE organisms grown in a growth medium
containing antibiotics will do this.
• Only GE organisms will grown in this
environment as the antibiotic will kill other
organisms that fail to take up the plasmid.
• This technique has worked well, but there
is concern about spreading “resistant
genes”.
Antibiotic resistance markers and
reporter systems
• Reporter systems have superseded antibiotic
resistant genes.
• This involves bioluminescence – target genes
will fluoresce, allowing them to be
followed/isolated
Another route to isolating a gene
• 20000-30000 protein coding genes in the human genome. Isolating
specific genes is difficult – complementary DNA technique is used
• Complementary DNA (cDNA) is copied from mRNA
• The enzyme reverse transcriptase synthesises DNA from an RNA template.
• Certain retroviruses have this enzyme capable of doing this.
• DNA produced is single stranded, but DNA polymerase can be used to
produce a double stranded cDNA “gene”.
• Beta cells in the pancreas make the hormone insulin, therefore
manufactures mRNA that codes for insulin. mRNA can be isolated and
used to make cDNA for the human insulin gene.
Another route to isolating a gene
• Advantages of GE using bacteria
– Microorganisms are simple and easy to
use
– Quick and easy to culture
– Few ethical issues about their use
• Disadvantages of GE using bacteria
– Cannot make human proteins
– Animals can be used, but products
have to be extracted via milk/urine and
not directly from cells
– Plants can be used – products secreted
via roots.