Download Ch5hybridisationSNPRFLP

Chapter 4: recombinant DNA
Restriction enzyme analysis
Cloning in E. coli plasmids
Transformation
Biomedical application
1
Restriction enzymes
• Restriction enzymes cut double-strand
DNA at specific recognition sequences
which are often 4-6 base pair palindromes
= 5’-3’ sequence is identical on both DNA
strands
• Many restriction enzymes cut the two DNA
strands at different points which
generates complementary single-strand
ends = sticky ends (others = blunt ends)
2
Restriction enzymes
BamHI (from B. amyloliquefaciens ) recognizes
GGATCC and cuts between the G’s on both strands
3
Restriction enzymes
4
Restriction enzymes
Restriction
enzymes
cut DNA
into defined
pieces,
named
restriction
fragments
5
Restriction enzymes
DNA fragments of different size (e.g.
restriction fragments) can be separated
according to their size by gel electrophoresis:
• agarose gel electrophoresis (300 bp - 15 kb)
• polyacrylamide gel electrophoresis (1-500 b)
=PAGE
6
Gel electrophoresis
Molecular weigth marker (band sizes
known) to compare sample band sizes
7
Recombinant DNA
• Sticky ends formed
by restriction enzymes
permit circularization or
combinations of DNA
restriction fragment(s)
by complementary
base pairing
8
Recombinant DNA
• A new combination of DNA can be made
by combining restriction fragments
• Complementary sticky ends can be
covalently linked with DNA ligase to form
recombinant DNA
• Blunt end DNA fragments (for example
generated by PCR) can also be ligated (but
less efficiently)
9
Recombinant DNA
Ligation of
vector and insert
DNA ligase
10
Recombinant DNA
• A vector is a replicating unit that can be
opened to insert another DNA fragment
• Often plasmids are used as vector in
bacteria
A plasmid is a small self-replicating circular
DNA molecule found in bacteria
11
Recombinant DNA
Plasmid vectors have
• an origin of replication
• a selectable marker gene (often an antibiotic
resistance)
• a cloning site or multicloning site (MCS)
12
Recombinant DNA
Transformation by heat shock or
electroshock
transformation
bacterium
Plasmid replication
Replicating
bacteria
form colony
13
Recombinant DNA
Selection
Plate bacteria on selective medium
R
Medium containing antibiotic
Select for presence of marker
14
Recombinant DNA
DNA
fragments
+
+
Vector
==
DNA 1
DNA 2
DNA 3
In reality only one or up
to millions of fragments
Recombinant DNA
molecules
15
Recombinant DNA Cloning = purification
Transform plasmids into bacteria: a cell will replicate
only one plasmid type
Plate bacteria to form colonies
16
Genetic engineering
Methods of genetic manipulation are named:
• Recombinant DNA technology
• Genetic engineering
• Gene cloning or gene technology
Applications include:
• Isolation of specific genes
• Production of specific proteins
17
Genetic engineering
• GMO = genetically modified organism,
GMM = genetically modified microorganisme
• Genetic modification = targeted
modification of a genetic characteristic
of an organism
•  transgenic organisme
Biomedical applications
• Recombinant DNA technology is used to
produce large amounts of medically
important proteins such as blood clotting
factors, insulin,…. In either bacteria, fungi,
animal cells, whole animals or plants
• DNA probes detect mutant genes in
hereditary diseases (DNA diagnostics)
19
Genetic engineering
• A chimeric gene is constructed of parts of
different genes
• An eukaryotic gene can only be expressed
in bacteria when provided with the correct
expression signals (and vice versa)
• Example: human insulin production in
bacteria
Bacterial promoter
Coding region human
insulin gene
Bacterial
terminator
20
Biomedical applications
• Diabetics lack the
hormone insulin
• Initially, insulin was
extracted from the
pancreas of cows or
pigs (different protein)
• Biotech insulin: safe
and easy