Download Molecular genetics (cloning)

Molecular genetics (cloning)
by
E. Börje Lindström
This learning object has been funded by the European Commissions FP6 BioMinE project
Definitions
• Genetic recombination:
- DNA from two different molecules
are combined – in vivo
- homology is necessary
• Recombinant DNA:
- combination of two DNA molecules in vitro
- complete homology is not necessary
• Molecular cloning:
- isolation of a bacterial population (a clone), that
all cell have a specific gene sequence.
Why cloning?
• The specific gene moved from a complex environment to a
less complex one
• easier to study
1) Structure and function of genes
2) Regulations
3) Sequencing of DNA
4) Production of
proteins/enzymes/ metabolites
Methodology
• The cloning can be divided into the following steps:
1) Selection of the gene to clone:
- isolation and fragmentation
2) Selection of a vector
3) Construction of a recombinant-molecule:
4) Introduction into a host cell
5) Selection of the ’right’ clone
6) Amplification
7) Analysis of the received DNA molecule
- chromosomal DNA/plasmid
10 Selection of the gene to clone
• Suppose that the gene is on a bacterial chromosome (genomic)
gene
chromosome

-isolate the chromosome
- cell debris
-fragment the chromosome

1
- enzymatic/ mechanically (shearing)
2
3
Restriction and Modification
• Foreign DNA entering the bacteria are
destroyed by:
• The own bacterial DNA is protected by:
- restriction enzymes/ endonucleases
- acting at specific sequences (4-6 bp)
-modification of the bases in these
sequences
- often through methylation (CH3-)
Some properties of these
enzymes
Restriction enzymes:
-cut within the DNA-molecule
- cut ds-DNA
- perpendicularly (straight) or
- symmetrically around the midpoint
- works on any type of DNA
- normally 1-5 ’cuts’ per DNAmolecule
Some properties of these
enzymes, cont.
Modification enzymes:
-the mehylases recognise the same sequence as its
equivalent restriction enzyme
- the methylase travel along the DNA-molecule with a
speed of ~ 150 bp/min
- must act perfectly otherwise the cell will dye
Summary:
• Important for the cell:
-protect its own DNA
- destroy foreign DNA
• Can be used in cloning of genes and analysis of
DNA-fragments
• There are several different restriction-modification systems in E. coli
20 Selection of a vector
• Viruses:
- e.g. l- phage
• Cosmids:
- the cos-sites of the l- phage
• Plasmids:
- Some properties that make plasmids good candidates
- Low molecular weight –easy to transfer
- Replicate independent of the chromosome
- Several copies/ cell
- Closed molecules  stableeasy to isolate
- Often only 1 restr. site/ enzyme
- Have selection traits (antibiotic resistance)
Where is the vector stored?
• Normally the vector (plasmid) is stored in the
bacterial cell!
Chromosome
Vector/plasmids

- isolation of the vector
- linearization with restriction enzymes
30 Construction of a recombinantmolecule
• In vitro
Genomic DNA

-use the same restriction enzyme for the
genomic DNA and the vector
- here BamH1
+ BamH1
Apr
Tcr
1
Plasmid
BamH1

+ BamH1
2
3
½ Tc
Apr
Mix + DNA ligase
½ Tc
30 Construction of a recombinantmolecule, cont.vector
1
½ Tc
2
Apr
½ Tc
3
Tcr
1
2
3
4
Apr
Apr
Apr
Apr
Re-ligated vector
0
4 Introduction
• Mechanism:
into a host cell
-Transformation (free recombinant-DNA)
- (Electroporation)
1
Apr
2
Apr
Tcr
3
Apr
4
Apr
40 Introduction into a host cell,
cont.
• Conclusion:
-The original chromosome with the special gene is distributed
into small pieces in the bacterial clone.
- The special gene is always in some of the bacteria
(a transformant)
50 How to find the bacterium
with the wanted gene?
• Cultivate the transformed culture
• Spread the culture on a nutrient plate and
select for transformants!
Replica plating
Nutrient agar
+ ampicillin

Nutrient agar
+ ampicillin +
tetracycline
0
5
How to find the bacterium
with the wanted gene, cont.?
• Among those not growing on tetracycline are
those with the gene inserted!
• How to find?
1) Suppose that the gene is expressed  new property
- Selection is possible
50 How to find the bacterium
with the wanted gene, cont.?
2) The gene is not expressed (no product)
- use some types of ’probes’
(radioactive, antibodies or stained)
- screening necessary
Colony/ plaque hybridization
Transfer
- lyse the bacteria


- denature the DNA
- add the probe
- wash out unbound probe
Master plate
Filter paper


- put the filter on a X-ray film
-The searched colony!
- Pick on master plate!
60 Amplification
• The copy number can be increase by blocking the
protein synthesis
• Through PCR (polymerase chain reaction)