An extensive English summary of the course Fundamentals of Gene and Genome Technology followed in academic year . Obtained result with this summary was 18/20. The explanations during the class were attentively noted and processed with the slides and course material to a complete summary. The summar...
CELL-BASED DNA CLONING
Principles of DNA cloning
- Cloning is a technique that is used in all labs over the world; it can be used for gene editing,
gene therapy, …
- 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
- Requires cutting and pasting of DNA
o Scissors: restriction endonucleases
o Glue: DNA ligase
- Requires also a replicon
o = a piece of DNA that makes independent DNA replication possible
o specific for a host
o usually a construct called ‘vector’ is used, containing many features for the cloning
process
à fragments of DNA cut with a restriction enzyme are mixed with a homogeneous population of
vector molecules that have been cut with the same RE
à target DNA and vector are joined by DNA ligase à recombinant DNA
! each vector contains an ORI: allow it to be copied when inside the cell
1
,2. transformation
- recombinant DNA molecule is introduced in a host cell
o usually bacterium or yeast: easy to grown, fast reproduction
o when the host cell divides, it will also replicate your recombinant DNA
- expression studies – cloning is often done in eukaryotic cells: mammalian cells, insect cells, …
o BUT: cloning in bacteria usually precedes cloning in the host used for the expression
à recombinant DNA is mixed with the host cells, which will pick up one foreign DNA
3. selective propagation of clones /amplification
- Cells are plated on agar (kind of sugar in a petri dish to grow bacteria)
- Each individual cell forms a colony – each colony is a clone à all cells are identical and have
the same ancestor cell
- A colony can be picked and can be grown in liquid medium to obtain more cells
à give a colony of identical cell clones containing one type of recombinant DNA
2
,4. Isolation of recombinant DNA clones
- the recombinant DNA is purified from the cells à lyse the cells and extract the recombinant
DNA
Restriction endonucleases (R.E.)
- tool we need for cloning
- nomenclature: 1 letter genus, 2 letters species, followed by number
o Example: HaeIII à Hemophilus aegypticus
- Isolated from bacteria, who have this as a defence mechanism against bacteriophages
(instead of an immune system in humans)
o Can cleave the viral DNA
o Bacteria also have a matching sequence specific DNA methylase
§ Methylation of the same recognition sequence in the bacteria
§ Protection: bacterial DNA cannot be cut
- Cut a certain sequence, very reliable tool
- Sequence that is cut: usually 4-8 bp, usually palindrome
cleavage
a. On the symmetry axis à blunt ends (no single stranded pieces)
b. Not on the symmetry axis à overhangs / sticky ends / cohesive termini
- Sticky ends can base pair and form unstable double helices
o 5’ overhang
o 3’ overhang
- isoschizomeres: different restriction enzymes that have the same recognition sequence
- example of a palindrome
- restriction endonuclease cuts between the two G’s à double stranded break
o result: single stranded part = overhang
3
, - DNA ligase can glue it back together: fosfodiester binding between the twe G’s à original
piece of DNA
Examples
- MboI and BamHI have compatible cohesive termini
o Both give a GATC overhang, although they recognize a different sequence
o So if the vector is cleaved with MboI and the DNA is cleaved with BamHI they can be
ligate together with DNA ligase
! SO: you don’t need always the same restriction enzymes to ligate them together
- Expected size of the fragments depends on the recognition sequence
o Correlation: the ones that give smaller pieces recognize 4 bases
- CpG islands have also an effect on the size
o CpG islands are methylated in human DNA for epigenetics
o If the enzyme recognizes CpG islands, the pieces will be longer
4
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