Gene and genome technology
Chapter 1: Cell-based DNA cloning
= cloning in a bacterium and multiply the DNA with the PCR (= amplify the DNA tube while
not amplify it in a cell, not every DNA will undergo this cell-based DNA that is still used).
Principles of DNA cloning
The cell-based DNA cloning comprises 4 steps:
1. In vitro construction of a recombinant DNA molecule (cutting and pasting DNA à
making recombine DNA from 2 different DNA origin human and bacteria
à this is an important step (cloning) in E. coli after that it can be put in a eukaryotic
cell for transformation
2. Transformation (in the host cell (= bacteria)) à grow it à multiply
3. Selective propagation of clones
4. Isolation of recombinant DNA clones (clear solution of the recombined DNA)
In vitro construction of a recombinant DNA molecule
• Requires cutting and pasting of DNA
o Restriction endonucleases
o DNA ligase
• Requires a replicon
o A piece of DNA that makes independent DNA replication possible (it will not
integrate into the chromosome bc it needs a lot of replication processes)
o Replicon is specific for a host
o Usually a construct called “vector” (often plasmid or phage) is used, containing
many features used in the cloning process (premade vectors are available by
research papers or companies).
1
,Transformation
• Recombinant DNA molecule is introduced in a host cell
• Usually bacterium or yeast
o Easy to grow (test tube + medium (37°C) à shake à growth after some
hours)
o Fast reproduction
• For expression studies, cloning is often done in eukaryotic cells (mammalian cells,
insect cells) à aren’t often used
• For expression studies, cloning in a bacterium usually precedes (=voorafgaand)
cloning in the host used for the expression
Selective propagation of clones
• Cells are plated on agar (also for bacteria)
• Each individual cell forms a colony
• Each colony is a clone: (small number of bacteria will be in dependable and grow in
dependable)
o All cells are identical, and have the same ancestor cell
• 1 colony is picked and can be grown in liquid medium to obtain more cells (selective
growth for the specific recombined dna (it will grow), if this doesn’t contain it (it won’t
grow)
Growth is
exponentyionally
à extracted the
plasmide and have
the recombine
DNA
2
,Isolation of recombinant DNA clones
The recombinant DNA is purified from the cells à lyse the cells and extract the recombinant
DNA
Example: an ion channel and introduce a mutation contain a region for the ion channel and
see if it changes the ion channel sequence.
à depending on the vector!
Restriction endonucleases
• Nomenclature: 1 letter genus, 2 letters species, followed by number
E.g. HaeIII: Hemophilus aegypticus (bacteria and III is the 3rd enzyme found in the
organism)
• Defense mechanism against bacteriophages (uses RE against phages)
o DNA from bacteriophage enters the bacterium and is cleaved
o There is a matching sequence specific DNA methylase
§ Methylation of the same recognition sequence (when this happens this
won’t be cut and the unmethylated seq phage will be cut.
§ Bacterial DNA cannot be cut (only the phage DNA (no infection of the
bacteria DNA and survives)
o Type II RE will cut a specific recognition sequence (known sequence)
o Usually 4-8 bp
o Usually palindrome (herkennings sequence: sequence dat hetzelfde zowel
voor als achter is (ex: ATTA))
Starts in Hemophilus aegypticus à transfer DNA in E. coli à works better for growth à
have RE (always cut the same seq and not the others).
3
, • Cleavage
o On the symmetry axis (= middle of the palindrome): blunt ends (cuts right in
the middle)
o Not on the symmetry axis: overhangs, sticky ends, cohesive termini
o Sticky ends can base pair and form unstable double helices
§ 5’ overhang
§ 3’ overhang
RE: (will always create a palindrome) is just cutting a strand of DNA, this side will not
contain phosphate, the other side will contain phosphate. This is because it only
contains one base (the side not contain phosphate).
DNA ligase joins the pieces together and construct the original DNA à recreate the RE
but it will be with other molecules. phosphodiester binding between the two G’s forms
the original piece of DNA
The 5’ prime overhang contains 4 bases while the 3’ prime overhang only has one
base.
4
Chapter 1: Cell-based DNA cloning
= cloning in a bacterium and multiply the DNA with the PCR (= amplify the DNA tube while
not amplify it in a cell, not every DNA will undergo this cell-based DNA that is still used).
Principles of DNA cloning
The cell-based DNA cloning comprises 4 steps:
1. In vitro construction of a recombinant DNA molecule (cutting and pasting DNA à
making recombine DNA from 2 different DNA origin human and bacteria
à this is an important step (cloning) in E. coli after that it can be put in a eukaryotic
cell for transformation
2. Transformation (in the host cell (= bacteria)) à grow it à multiply
3. Selective propagation of clones
4. Isolation of recombinant DNA clones (clear solution of the recombined DNA)
In vitro construction of a recombinant DNA molecule
• Requires cutting and pasting of DNA
o Restriction endonucleases
o DNA ligase
• Requires a replicon
o A piece of DNA that makes independent DNA replication possible (it will not
integrate into the chromosome bc it needs a lot of replication processes)
o Replicon is specific for a host
o Usually a construct called “vector” (often plasmid or phage) is used, containing
many features used in the cloning process (premade vectors are available by
research papers or companies).
1
,Transformation
• Recombinant DNA molecule is introduced in a host cell
• Usually bacterium or yeast
o Easy to grow (test tube + medium (37°C) à shake à growth after some
hours)
o Fast reproduction
• For expression studies, cloning is often done in eukaryotic cells (mammalian cells,
insect cells) à aren’t often used
• For expression studies, cloning in a bacterium usually precedes (=voorafgaand)
cloning in the host used for the expression
Selective propagation of clones
• Cells are plated on agar (also for bacteria)
• Each individual cell forms a colony
• Each colony is a clone: (small number of bacteria will be in dependable and grow in
dependable)
o All cells are identical, and have the same ancestor cell
• 1 colony is picked and can be grown in liquid medium to obtain more cells (selective
growth for the specific recombined dna (it will grow), if this doesn’t contain it (it won’t
grow)
Growth is
exponentyionally
à extracted the
plasmide and have
the recombine
DNA
2
,Isolation of recombinant DNA clones
The recombinant DNA is purified from the cells à lyse the cells and extract the recombinant
DNA
Example: an ion channel and introduce a mutation contain a region for the ion channel and
see if it changes the ion channel sequence.
à depending on the vector!
Restriction endonucleases
• Nomenclature: 1 letter genus, 2 letters species, followed by number
E.g. HaeIII: Hemophilus aegypticus (bacteria and III is the 3rd enzyme found in the
organism)
• Defense mechanism against bacteriophages (uses RE against phages)
o DNA from bacteriophage enters the bacterium and is cleaved
o There is a matching sequence specific DNA methylase
§ Methylation of the same recognition sequence (when this happens this
won’t be cut and the unmethylated seq phage will be cut.
§ Bacterial DNA cannot be cut (only the phage DNA (no infection of the
bacteria DNA and survives)
o Type II RE will cut a specific recognition sequence (known sequence)
o Usually 4-8 bp
o Usually palindrome (herkennings sequence: sequence dat hetzelfde zowel
voor als achter is (ex: ATTA))
Starts in Hemophilus aegypticus à transfer DNA in E. coli à works better for growth à
have RE (always cut the same seq and not the others).
3
, • Cleavage
o On the symmetry axis (= middle of the palindrome): blunt ends (cuts right in
the middle)
o Not on the symmetry axis: overhangs, sticky ends, cohesive termini
o Sticky ends can base pair and form unstable double helices
§ 5’ overhang
§ 3’ overhang
RE: (will always create a palindrome) is just cutting a strand of DNA, this side will not
contain phosphate, the other side will contain phosphate. This is because it only
contains one base (the side not contain phosphate).
DNA ligase joins the pieces together and construct the original DNA à recreate the RE
but it will be with other molecules. phosphodiester binding between the two G’s forms
the original piece of DNA
The 5’ prime overhang contains 4 bases while the 3’ prime overhang only has one
base.
4
