Genome Technology & Applications
Chapter 1: Cell-based DNA cloning: 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
− 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
o Replicon is specific for a host
o Usually a construct called “vector” is used, containing many features
o used in the cloning process.
→ 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
→ target DNA and vector are joined by DNA
ligase → recombinant DNA ! each vector
contains an ORI: allow it to be copied when
inside the cell
2. Transformation
− Recombinant DNA molecule is introduced in a host cell
− Usually bacterium or yeast
o Easy to grown
o Fast reproduction
− For expression studies, cloning is often done in eukaryotic cells (mammalian cells, insect cells, see
later in this chapter)
− For expression studies, cloning in a bacterium usually precedes cloning in the host used for the
expression
→recombinant DNA is mixed with the host
cells, which will pick up one foreign DNA
1
,3. Selective propagation of clones
− 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
→ give a colony of identical cell
clones containing one type of
recombinant DNA
4. Isolation of recombinant DNA clones
The recombinant DNA is purified firm the cells → lyse the cells and extract the recombinant DNA
5. Restriction endonucleases
− Tool we need for cloning
− Nomenclature: 1 letter genus, 2 letters species, followed by number
E.g. HaeIII: Hemophilus aegypticus
− Defence mechanism against bacteriophages
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
▪ Bacterial DNA cannot be cut
o Type II RE will cut a specific recognition sequence, very reliable tool
o Usually 4-8 bp
o Usually palindrome
2
,5.1 Cleavage
− On the symmetry axis → blunt ends (no single stranded pieces)
− Not on the symmetry axis → 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 → double stranded break
Result: single stranded part = overhang
DNA ligase can glue it back together: fosfodiester binding between the twe G’s → original piece
of DNA
− Some RE have compatible cohesive termini
E.g. BamHI: G↓GATCC en MboI: ↓GATC
Both give a GATC overhang, although they recognize a different sequence. So if the vector is
cleaved with MboI and the DNA is cleaved with BamHI they can be ligate together with DNA
ligase.
− 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
! a methylated C will not be repaired as easy as an unmethylated
− It is also known that the human genome consists more of A and T then G and C
o More G and C = longer fragment
3
, 6. DNA ligase
− Tool that we need for cloning
− DNA ligase can restore a covalent
bond in a DNA molecule
− Sticky ends that are hybridised
together are instable, even at low
temperature
− The hydrogen bonds of the double
helix facilitate the work of DNA ligase
o A-T: 2 H-bridges
o G-C: 3 H-bridges
− aim: intermolecular recombinant DNA
that is partly vector and partly DNA
o there are strategies to avoid
concatamere and vector
cyclization (see later)
− Several different fragments that are
ligated together are called a
concatamere
o not necessary the same as the original
o you can recreate a genome
− Intramolecular ligation is called vector cyclisation
7. Origin of replication (ORI)
− Another tool
− An ORI allows replication independent of the host chromosome and limits the number of
chromosomes to 1 per cell
− Independent replication facilitates purification of recombinant molecule
Bacterial chromosome:
− Circular chromosome with 1 ORI
! plasmids have another type of ORI
4
Chapter 1: Cell-based DNA cloning: 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
− 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
o Replicon is specific for a host
o Usually a construct called “vector” is used, containing many features
o used in the cloning process.
→ 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
→ target DNA and vector are joined by DNA
ligase → recombinant DNA ! each vector
contains an ORI: allow it to be copied when
inside the cell
2. Transformation
− Recombinant DNA molecule is introduced in a host cell
− Usually bacterium or yeast
o Easy to grown
o Fast reproduction
− For expression studies, cloning is often done in eukaryotic cells (mammalian cells, insect cells, see
later in this chapter)
− For expression studies, cloning in a bacterium usually precedes cloning in the host used for the
expression
→recombinant DNA is mixed with the host
cells, which will pick up one foreign DNA
1
,3. Selective propagation of clones
− 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
→ give a colony of identical cell
clones containing one type of
recombinant DNA
4. Isolation of recombinant DNA clones
The recombinant DNA is purified firm the cells → lyse the cells and extract the recombinant DNA
5. Restriction endonucleases
− Tool we need for cloning
− Nomenclature: 1 letter genus, 2 letters species, followed by number
E.g. HaeIII: Hemophilus aegypticus
− Defence mechanism against bacteriophages
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
▪ Bacterial DNA cannot be cut
o Type II RE will cut a specific recognition sequence, very reliable tool
o Usually 4-8 bp
o Usually palindrome
2
,5.1 Cleavage
− On the symmetry axis → blunt ends (no single stranded pieces)
− Not on the symmetry axis → 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 → double stranded break
Result: single stranded part = overhang
DNA ligase can glue it back together: fosfodiester binding between the twe G’s → original piece
of DNA
− Some RE have compatible cohesive termini
E.g. BamHI: G↓GATCC en MboI: ↓GATC
Both give a GATC overhang, although they recognize a different sequence. So if the vector is
cleaved with MboI and the DNA is cleaved with BamHI they can be ligate together with DNA
ligase.
− 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
! a methylated C will not be repaired as easy as an unmethylated
− It is also known that the human genome consists more of A and T then G and C
o More G and C = longer fragment
3
, 6. DNA ligase
− Tool that we need for cloning
− DNA ligase can restore a covalent
bond in a DNA molecule
− Sticky ends that are hybridised
together are instable, even at low
temperature
− The hydrogen bonds of the double
helix facilitate the work of DNA ligase
o A-T: 2 H-bridges
o G-C: 3 H-bridges
− aim: intermolecular recombinant DNA
that is partly vector and partly DNA
o there are strategies to avoid
concatamere and vector
cyclization (see later)
− Several different fragments that are
ligated together are called a
concatamere
o not necessary the same as the original
o you can recreate a genome
− Intramolecular ligation is called vector cyclisation
7. Origin of replication (ORI)
− Another tool
− An ORI allows replication independent of the host chromosome and limits the number of
chromosomes to 1 per cell
− Independent replication facilitates purification of recombinant molecule
Bacterial chromosome:
− Circular chromosome with 1 ORI
! plasmids have another type of ORI
4
