General info
- 4 professors
- Seminar on Next Genera3on Sequencing
o Prepara3on and a:endance is compulsory
o In small groups
o Prepare a small 10 minute presenta3on on a certain topic related to NGS
o Different topics foreseen
o Follow-up seminar with discussion
- Slides on BB
Cell-based DNA cloning
Principles of DNA cloning
- Developed in the 1970s
- Used a lot and also today
- 4 steps
- Early: cloning was used to amplify DNA. There was no PCR un3l the late 80s
1. In vitro construc.on of a recombinant DNA molecule
- Requires cuRng and pas3ng of DNA
o Restric3on endonucleases (cuRng)
o DNA ligase (pas3ng)
- Requires a replicon (= piece of DNA that makes independent replica3on possible )
o A piece of DNA that makes independent DNA replica3on possible
o Replicon is specific for a host
o Usually a construct called “vector” is used, containing many features used in the cloning
process.
§ A vector: usually a plasmid, but has engineered -> specific features that makes it
suitable
2. Transforma.on in a host (o<en a bacteria -> will replicate the DNA)
- Recombinant DNA molecule is introduced in a host cell
- Usually bacterium or yeast
o Easy to grown
o Fast reproduc3on
- For expression studies, cloning is o]en done in eukaryo3c cells (mammalian cells, insect cells,
see later in this chapter)
o For some research this is important. You bring your recombinant molecule in the
mammalian cells and you can study the cells.
- For expression studies, cloning in a bacterium usually precedes cloning in the host used for
the expression
o Cloning in bacteria will always need to be done. Before puRng the construct in the
mammalian cells you will make the construct in bacteria -> the finished product will be
cloned in mammalian cells
, 3. Selec.ve propaga.on of clones
- Cells are plated on agar
- Each individual cell forms a colony
- Each colony is a clone:
o All cells are iden3cal, and have the same ancestor cell
- 1 colony can be grown in liquid medium to obtain more cells
- A]er the transforma3on you can bring the bacteria on agar -> grown and form colony (all
cells in the colony are iden3cal). You can take one colony and growth it in liquid media
4. Isola.on of recombinant DNA clones
- The recombinant DNA is purified from the cells
- You can take out the construct a]er cloning
o You take one colony -> you put in a liquid medium -> growing it overnight at 37°C -> lyse
and purify
Further into it
1. Restric.on endonucleases
- CuRng
- Nomenclature: 1 le:er genus, 2 le:ers species, followed by number
o E.g. HaeIII: Hemophilus aegyp3cus
- We get our restric3on enzymes from bacteria
o Natural defence mechanism against bacteriophages
o DNA from bacteriophage enters the bacterium then it is cleaved
o There is a matching sequence specific DNA methylase
§ Methyla3on of the same recogni3on sequence
§ Bacterial DNA cannot be cut
è Restric3on endonucleases recognizes a specific DNA sequence -> will only cut that
sequence. It will methylate its own DNA and it will make the restric3on endonucleases that
way that its own DNA is not cut
o Type II RE will cut a specific recogni3on sequence
o Usually 4-8 bp
o Usually palindrome
- Cleavage
o On the symmetry axis: blunt ends
o Not on the symmetry axis : overhangs, s3cky ends, cohesive termini
- 4 professors
- Seminar on Next Genera3on Sequencing
o Prepara3on and a:endance is compulsory
o In small groups
o Prepare a small 10 minute presenta3on on a certain topic related to NGS
o Different topics foreseen
o Follow-up seminar with discussion
- Slides on BB
Cell-based DNA cloning
Principles of DNA cloning
- Developed in the 1970s
- Used a lot and also today
- 4 steps
- Early: cloning was used to amplify DNA. There was no PCR un3l the late 80s
1. In vitro construc.on of a recombinant DNA molecule
- Requires cuRng and pas3ng of DNA
o Restric3on endonucleases (cuRng)
o DNA ligase (pas3ng)
- Requires a replicon (= piece of DNA that makes independent replica3on possible )
o A piece of DNA that makes independent DNA replica3on possible
o Replicon is specific for a host
o Usually a construct called “vector” is used, containing many features used in the cloning
process.
§ A vector: usually a plasmid, but has engineered -> specific features that makes it
suitable
2. Transforma.on in a host (o<en a bacteria -> will replicate the DNA)
- Recombinant DNA molecule is introduced in a host cell
- Usually bacterium or yeast
o Easy to grown
o Fast reproduc3on
- For expression studies, cloning is o]en done in eukaryo3c cells (mammalian cells, insect cells,
see later in this chapter)
o For some research this is important. You bring your recombinant molecule in the
mammalian cells and you can study the cells.
- For expression studies, cloning in a bacterium usually precedes cloning in the host used for
the expression
o Cloning in bacteria will always need to be done. Before puRng the construct in the
mammalian cells you will make the construct in bacteria -> the finished product will be
cloned in mammalian cells
, 3. Selec.ve propaga.on of clones
- Cells are plated on agar
- Each individual cell forms a colony
- Each colony is a clone:
o All cells are iden3cal, and have the same ancestor cell
- 1 colony can be grown in liquid medium to obtain more cells
- A]er the transforma3on you can bring the bacteria on agar -> grown and form colony (all
cells in the colony are iden3cal). You can take one colony and growth it in liquid media
4. Isola.on of recombinant DNA clones
- The recombinant DNA is purified from the cells
- You can take out the construct a]er cloning
o You take one colony -> you put in a liquid medium -> growing it overnight at 37°C -> lyse
and purify
Further into it
1. Restric.on endonucleases
- CuRng
- Nomenclature: 1 le:er genus, 2 le:ers species, followed by number
o E.g. HaeIII: Hemophilus aegyp3cus
- We get our restric3on enzymes from bacteria
o Natural defence mechanism against bacteriophages
o DNA from bacteriophage enters the bacterium then it is cleaved
o There is a matching sequence specific DNA methylase
§ Methyla3on of the same recogni3on sequence
§ Bacterial DNA cannot be cut
è Restric3on endonucleases recognizes a specific DNA sequence -> will only cut that
sequence. It will methylate its own DNA and it will make the restric3on endonucleases that
way that its own DNA is not cut
o Type II RE will cut a specific recogni3on sequence
o Usually 4-8 bp
o Usually palindrome
- Cleavage
o On the symmetry axis: blunt ends
o Not on the symmetry axis : overhangs, s3cky ends, cohesive termini
