This is a full summary of all lectures of the course 'Genome Technology and Applications', given in the first Master year of Biomedical Sciences. This includes all lectures given by professors Guy van Camp and Wim Van Hul. For the lectures of professor Frank Kooy and NGS techniques, there is a sepa...
Genome technology and
applications
CELL-BASED DNA CLONING 29/9
= when you clone in cells. Sometimes techniques like PCR are called in vitro cloning. Cloning means
amplifying nucleic acids.
PRINCIPLES OF DNA CLONING
Cell based DNA cloning comprises 4 steps:
1. In vitro construction of a recombinant DNA molecule
2. Transformation
3. Selective propagation of clones
4. Isolation of recombinant DNA clones
1. IN VITRO CONSTRUCTION OF A RECOMBINANT DNA MOLECULE
This is always done in vitro. It requires cutting and pasting of DNA by enzymes:
- Resitriction endonucleases: cut DNA
- DNA ligase: seals cuts.
To make a recombinant DNA molecule, we require a replicon. This is a piece of DNA that makes
independent DNA replication possible.
- Replicon is specific for a host
- Usually a construct called “vector” is used
o = piece of DNA containing the replicon and many elements that make the cloning
process possible
In most cases an ORI is needed: origin of replication, a DNA sequence that makes a DNA polymerase
work. If the cell divides, extra DNA of the recombinant DNA molecule is made and is passed on to
daughter cells. If you don’t have an ORI, it will not be replicated and the recombinant DNA will be
lost.
,Plasmids are usually used in cloning as a vector. You cut the plasmid and target DNA open with the
same restriction enzyme after which you can ligate the target DNA in a recombinant DNA molecule.
2. TRANSFORMATION
The recombinant DNA molecule is introduced in a host cell. This is usually bacterium or yeast but can
also be eukaryotic cells. Bacteria and yeasts are very popular because they’re easy to grown and have
a fast reproduction.
For expression studies, cloning is often done in eukaryotic cells (mammalian cells, insect cells, see
later in this chapter). If you want to express human proteins you will need eukaryotic cells because
they’re often too big for bacteria. You will often still begin in cloning in a bacterium. Next, you put it
in the eukaryotic cells for the expression.
3. SELECTIVE PROPAGATION OF CLONES
After transformation, the cells are plated on agar. Each individual cell forms a colony. Each colony is a
clone: All cells are identical, and have the same ancestor cell. 1 colony can be grown in liquid medium
to obtain more cells. Usually the vector contains an antibiotic resistance genes, and the agar contains
this antibiotic, so only the transformed bacteria can survive.
4. ISOLATION OF RECOMBINANT DNA CLONES
If you want to isolate your construct and multiply it, you have to lyse the bacteria and purify the
plasmid: the recombinant DNA is purified from the cells. This is pretty easy since the bacterial
genome is bigger.
, E.g. Hemophilus
RESTRICTION ENDONUCLEASES aegypticus
HaeIII
Nomenclature: 1 letter genus + 2 letters species + followed by number
Restriction endonucleases (RE) are a defence mechanism against bacteriophages. Bacteriophage DNA
enters the bacterium and is cleaved by RE. The bacteria has to make sure its own DNA is not cut.
That’s why a RE always has a matching methylase. This methylation will be of the same recognition
sequence. In bacteria, all sequences are methylated and restriction endonucleases never cut
methylated DNA.
RE are very specific and never make mistakes. The sequence that they recognize is 4-8 bp and is
usually a palindrome. RE can cut on the symmetry axis, but can also cut somewhere else. In this case,
it creates overhanging sticky ends which can base pair and form unstable double helices.
5’ prime overhang
The overhangs are complimentary so they can base pair. They’re not stable at room temperature but
can hybridize shortly. The short hybridization makes it easier to ligate the cut again. DNA ligase can
also ligate blunt ends, but it’s more difficult.
Some restriction enzyme give long DNA pieces and others give short pieces:
- Depending on length of recognition sequences
o Shorter recognition sequences more present shorter pieces
o Longer recognition sequences less present longer pieces
- The human genome has only 40% GC’s and 60% AT’s
o Recognition sequence with only G and C’s less present longer pieces
- CPG sequence (-CG-): site where methylation happens. Methylated C’s mutate away more
often to T’s so there aren’t that many.
o Recognition sequence with -CG- in it less present long pieces
Isoschizomeres: when different restriction enzymes have the same recognition sequence.
Some RE have compatible cohesive termini. After a cut they have compatible ends and can be ligated
DNA LIGASE
, - Can restore a covalent bond in a DNA molecule
- Sticky ends that are hybridised together are instable
o Even at low temperature
- Hydrogen bonds of the double helix facilitate ligation
- Different fragments that are ligated together = concatamere
o E.g. pathwork of chromosomes
o You want 1 insert in 1 vector so this is not wanted
When cut open, the plasmid has 2 compatible ends. It’s possible
that the vector is closed without DNA entering. If you don’t add
a lot of DNA to the ‘mixture’ this is very likely to happen. This
intramolecular ligation is called cyclisation.
CHARACTERISTICS OF THE VECTOR
ORIGIN OF REPLICATION (ORI)
The ORI allows replication independent of the host chromosome. This is needed so you don’t lose the
recombinant DNA molecule as the cell divided.
Most bacteria have a circular chromosomes with one ORI which limits the number of chromosomes
of the cell to 1. If plasmids are present, more than one can be present. The ORI of plasmids is
different: if bacteria would divide and there is only 1 plasmid then only one daughter cell will have
the plasmid.
MOST USED VECTORS
Plasmids
- Small circular DNA molecules in bacteria
- Usually multiple copy number ORI: to make sure there are enough plasmids
- Usually contain only a few genes
- Transmitted vertically from parental to daughter cells
- Transmitted horizontally from one bacterium to another via conjugation
- Have a supercoil structure (same as bacterial chromosome)
o This makes the plasmid stable against DNA denaturation
o Easiest way to prepare plasmids: boiling bacteria gives quite pure plasmid DNA
Bacteriophages are viruses that infect bacteria. They contain a linear or circular genome and can be
found outside the cell in a protein coat, which makes isolating DNA quite easy.
AVOIDING RECIRCULARIZATION IN VECTORS
1. USING TWO DIFFERENT RESTRICTION ENZYMES
When cloning, you want to avoid recircularization of the vector. You can do
this by using two RE with incompatible ends: e.g. EcoRI and HindIII. Many
vectors have a multiple cloning site: a place where you can do cloning and
there are several RE sites. When you cut the vector and the insertion with EcoRI
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