Genome technology and applications
Cell-based DNA cloning:
1. In vitro construction of a recombinant DNA molecule:
Requirements:
Restriction endonucleases (“scissors”)
DNA ligase (“glue”)
Replicon: DNA that makes independent DNA replication possible
o Often vectors used (in this case plasmids)
o Replicons are specific per host
Restriction endonuclease:
Restriction endonucleases: bacteria’s defence mechanism against bacteriophages
Contains a recognition sequence: this same sequence is methylated in the bacterium so
won’t be cut , viral DNA will be cleaved
o 4-8 bp palindrome
Bacteriophages: viruses infecting bacteria with a linear/circular genome
Recognition sequence: size of cleavage product increases with increased length of the recognition sequence
because it is more rare in the genome bv AluI has AGTC so expected size is 300bp (short)
If sequence has GC also longer expected sequence due to CpG methylation sites
Cleavage: can cause blunt ends(cut on symmetry axis) or sticky ends that can form base pairs
MboI and BamHI: produce the same overhanging ends
= Isoschizomeres
Compatible products so can be ligated
Nomenclature: 1 letter genus, 2 letter species, number bv HaeIII = 3rd restriction enzyme of Hemophilus aegypticus
Avoid recircularization:
2 restriction enzymes: cut both the vector and insertion with different 2 RE so it
cannot ligate with itself
Dephosphorylation: alkaine phosphatase removes the 5’ phosphate so the vector cannot
recircularize (DNA ligase needs 5’ phosphate group and 3’ OH-group)
DNA insert still has phosphate group so can ligate with the vector
,DNA ligase:
DNA ligase: restores covalent bond in a DNA molecule
Sticky ends: are unstable if only hybridized together so DNA ligase will stabilize these
Hydrogen bonds in double helix facilitate DNA ligase
Ligation products: are either
Intermolecular target-target concatemers
Intramolecular vector cyclization
Intermolecular vector-target recombinant DNA
Concatemer: several different fragments ligated together
Cyclisation: intramolecular ligation
Vectors:
Origin of replication: allows replication independent of the host chromosome
needed for purification of the recombinant molecule
Plasmid: small circular bacterial DNA molecule
Multiple copy number ORI’s
Contains only a few genes
Supercoiled: additional twists cause molecule to coil up
Vertical transmission from parent to daughter cells / horizontal via conjugation to another bacterium
2. Transformation:
Transformation: uptake of DNA in the cell
Inefficient process in bacteria
Increase uptake efficiency:
Ca2+-buffer: makes competent cells that can take up DNA (better when combined with heat shock)
Electric shock: brief high voltage pulse makes holes in bacterial cell-wall and membrane (electroporation)
Circular DNA more easily transformed than linear
Host cell:
Eukaryotic cell: used in expression studies
Bacterium/ yeast: used for the preparation work for the expression study
o Easy to grow
o Fast reproduction
3. Selective propagation of clones:
1. Cells plated on agar
2. Cells grow and form colonies (all cells in 1 colony are identical (from same ancestor))
3. 1 colony isolated and grown in liquid medium (secondary amplification)
Recombinant screening:
,Recombinant screening: make sure bacteria contains the plasmid by only allowing it to grow if
plasmid is inserted
Antibiotic resistance: vector contains antibiotic resistance gene so when plating the bacteria on an
antibiotic coated agar plate only those containing vectors can grow
Vectors without inserts: differentiate between colonies with empty vectors and with inserts
β -galactosidase gene complementation
Supressor tRNA genes
β -galactosidase gene complementation:
β -galactosidase gene:
Host cell: contains an incomplete β -galactosidase gene missing the amino-terminal part
Vector: contains the amino-terminal part (LacZ gene)
2 protein fragments will form a functional protein
X-gal: colorless product converted into blue dye by β -galactosidase
Colonies containing the vector will turn blue
Insertional inactivation: in the LacZ gene there is a multiple cloning site that normally does not
disturb the gene (limited bases , multiple of 3)
Large insert: will interrupt the MCS and interrupt the LacZ gene
Colonies with an insertion are colorless
Supressor tRNA genes:
Supressor tRNA: tRNA with a mutation in the anticodon that causes it to recognise a stop-codon and
fill in an AA protein doesn’t get truncated
Host cell: is mutant for a premature stopcodon Recognisable bv color due to accumulation of a product
Vector: contains supressor tRNA with the multiple cloning site in the middle
No vector: coloured due to premature stopcodon mutation
Empty vector: white because intact supressor tRNA will insert AA at premature stopcodon site
Vector + insert: coloured due to supressor tRNA inactivation
4. Isolation of recombinant DNA clones:
DNA isolation: by cell lysis and purification to only isolate the plasmid containing the DNA sequence
Plasmid preparation in E. Coli:
E. Coli: has circular genome of 4,6Mb and plasmid of a few kb
1. Cell lysis: apply lysozyme to break cell wall
2. Chromosome gets broken in linear pieces during lysis , plasmid remains circular
3. Denature and renature DNA: increase pH to 12 and back to neutral
o Linear pieces: cannot renature quickly
o Circular pieces: renature perfectly because complementary strands remain close
together due to supercoil
4. Denatured DNA + proteins precipitate during the fast renaturation
5. Remaining solution is pure plasmid
, Cloning in bacteriophage lambda:
Bacteriophage lambda: linear dsDNA genome of 50kb packed in protein coat and tail
Cohesive sites: 5’ overhangs of 12bp that facilitate the transition of linear in the phage head to
circular in the cell (hybridises with itself to circularize)
Critical sequence for DNA packaging in phage heads
Lytic cycle: formation of virus particles
1. DNA replication
2. Synthesis of coat proteins
3. Packaging of phage DNA in coat proteins
4. Host cell lysis
Lysogenic cycle: viral DNA integrates in bacterial genome = provirus
by recombination at the att sequence
After a few generations of bacteria virus particles will be made
from the provirus
Lambda genome:
Integration and recombination genes: non-essential because lysogenic pathway not used in cloning
= stuffer fragment
In vitro lambda packaging:
Protein mix: packages lambda DNA in phage particles and consists of bacterial strains containing
lysogenic phages that are blocked in different steps of the packaging process
o 1 strain doesn’t have E protein (major capsid protein for preheads)
o 1 has protein E but misses protein D (for assembly)
1. Lyse both bacteria and mixing the lysates
2. Add DNA with cohesive sites
3. DNA will be packaged in the phages (if cos sites are 50kb apart)
Lambda cloning vectors:
Replacement vectors: stuffer fragment replaced by insert (max 20kb)
will only be long enough to package in phage heads with insert present (total DNA 37-50kb)
Used for genomic DNA
Insertion vectors: short vector with DNA of max 10kb
Used for cDNA
Cosmid vectors: hybrid between plasmid and phages by cloning a cos sequence in a plasmid
1. MboI cuts plasmid , BamHI cuts insert to create the same overhanging ends
2. Insert (max 30-45kb) ligated in the plasmid (use in vitro packaging mix)
3. Vector put in phase head using the cos sites
Combines plasmid convenience with phage efficiency
Fosmid: cosmid based on the F plasmid of E. Coli (lower copy number ORI – less DNA but more stable)
Cell-based DNA cloning:
1. In vitro construction of a recombinant DNA molecule:
Requirements:
Restriction endonucleases (“scissors”)
DNA ligase (“glue”)
Replicon: DNA that makes independent DNA replication possible
o Often vectors used (in this case plasmids)
o Replicons are specific per host
Restriction endonuclease:
Restriction endonucleases: bacteria’s defence mechanism against bacteriophages
Contains a recognition sequence: this same sequence is methylated in the bacterium so
won’t be cut , viral DNA will be cleaved
o 4-8 bp palindrome
Bacteriophages: viruses infecting bacteria with a linear/circular genome
Recognition sequence: size of cleavage product increases with increased length of the recognition sequence
because it is more rare in the genome bv AluI has AGTC so expected size is 300bp (short)
If sequence has GC also longer expected sequence due to CpG methylation sites
Cleavage: can cause blunt ends(cut on symmetry axis) or sticky ends that can form base pairs
MboI and BamHI: produce the same overhanging ends
= Isoschizomeres
Compatible products so can be ligated
Nomenclature: 1 letter genus, 2 letter species, number bv HaeIII = 3rd restriction enzyme of Hemophilus aegypticus
Avoid recircularization:
2 restriction enzymes: cut both the vector and insertion with different 2 RE so it
cannot ligate with itself
Dephosphorylation: alkaine phosphatase removes the 5’ phosphate so the vector cannot
recircularize (DNA ligase needs 5’ phosphate group and 3’ OH-group)
DNA insert still has phosphate group so can ligate with the vector
,DNA ligase:
DNA ligase: restores covalent bond in a DNA molecule
Sticky ends: are unstable if only hybridized together so DNA ligase will stabilize these
Hydrogen bonds in double helix facilitate DNA ligase
Ligation products: are either
Intermolecular target-target concatemers
Intramolecular vector cyclization
Intermolecular vector-target recombinant DNA
Concatemer: several different fragments ligated together
Cyclisation: intramolecular ligation
Vectors:
Origin of replication: allows replication independent of the host chromosome
needed for purification of the recombinant molecule
Plasmid: small circular bacterial DNA molecule
Multiple copy number ORI’s
Contains only a few genes
Supercoiled: additional twists cause molecule to coil up
Vertical transmission from parent to daughter cells / horizontal via conjugation to another bacterium
2. Transformation:
Transformation: uptake of DNA in the cell
Inefficient process in bacteria
Increase uptake efficiency:
Ca2+-buffer: makes competent cells that can take up DNA (better when combined with heat shock)
Electric shock: brief high voltage pulse makes holes in bacterial cell-wall and membrane (electroporation)
Circular DNA more easily transformed than linear
Host cell:
Eukaryotic cell: used in expression studies
Bacterium/ yeast: used for the preparation work for the expression study
o Easy to grow
o Fast reproduction
3. Selective propagation of clones:
1. Cells plated on agar
2. Cells grow and form colonies (all cells in 1 colony are identical (from same ancestor))
3. 1 colony isolated and grown in liquid medium (secondary amplification)
Recombinant screening:
,Recombinant screening: make sure bacteria contains the plasmid by only allowing it to grow if
plasmid is inserted
Antibiotic resistance: vector contains antibiotic resistance gene so when plating the bacteria on an
antibiotic coated agar plate only those containing vectors can grow
Vectors without inserts: differentiate between colonies with empty vectors and with inserts
β -galactosidase gene complementation
Supressor tRNA genes
β -galactosidase gene complementation:
β -galactosidase gene:
Host cell: contains an incomplete β -galactosidase gene missing the amino-terminal part
Vector: contains the amino-terminal part (LacZ gene)
2 protein fragments will form a functional protein
X-gal: colorless product converted into blue dye by β -galactosidase
Colonies containing the vector will turn blue
Insertional inactivation: in the LacZ gene there is a multiple cloning site that normally does not
disturb the gene (limited bases , multiple of 3)
Large insert: will interrupt the MCS and interrupt the LacZ gene
Colonies with an insertion are colorless
Supressor tRNA genes:
Supressor tRNA: tRNA with a mutation in the anticodon that causes it to recognise a stop-codon and
fill in an AA protein doesn’t get truncated
Host cell: is mutant for a premature stopcodon Recognisable bv color due to accumulation of a product
Vector: contains supressor tRNA with the multiple cloning site in the middle
No vector: coloured due to premature stopcodon mutation
Empty vector: white because intact supressor tRNA will insert AA at premature stopcodon site
Vector + insert: coloured due to supressor tRNA inactivation
4. Isolation of recombinant DNA clones:
DNA isolation: by cell lysis and purification to only isolate the plasmid containing the DNA sequence
Plasmid preparation in E. Coli:
E. Coli: has circular genome of 4,6Mb and plasmid of a few kb
1. Cell lysis: apply lysozyme to break cell wall
2. Chromosome gets broken in linear pieces during lysis , plasmid remains circular
3. Denature and renature DNA: increase pH to 12 and back to neutral
o Linear pieces: cannot renature quickly
o Circular pieces: renature perfectly because complementary strands remain close
together due to supercoil
4. Denatured DNA + proteins precipitate during the fast renaturation
5. Remaining solution is pure plasmid
, Cloning in bacteriophage lambda:
Bacteriophage lambda: linear dsDNA genome of 50kb packed in protein coat and tail
Cohesive sites: 5’ overhangs of 12bp that facilitate the transition of linear in the phage head to
circular in the cell (hybridises with itself to circularize)
Critical sequence for DNA packaging in phage heads
Lytic cycle: formation of virus particles
1. DNA replication
2. Synthesis of coat proteins
3. Packaging of phage DNA in coat proteins
4. Host cell lysis
Lysogenic cycle: viral DNA integrates in bacterial genome = provirus
by recombination at the att sequence
After a few generations of bacteria virus particles will be made
from the provirus
Lambda genome:
Integration and recombination genes: non-essential because lysogenic pathway not used in cloning
= stuffer fragment
In vitro lambda packaging:
Protein mix: packages lambda DNA in phage particles and consists of bacterial strains containing
lysogenic phages that are blocked in different steps of the packaging process
o 1 strain doesn’t have E protein (major capsid protein for preheads)
o 1 has protein E but misses protein D (for assembly)
1. Lyse both bacteria and mixing the lysates
2. Add DNA with cohesive sites
3. DNA will be packaged in the phages (if cos sites are 50kb apart)
Lambda cloning vectors:
Replacement vectors: stuffer fragment replaced by insert (max 20kb)
will only be long enough to package in phage heads with insert present (total DNA 37-50kb)
Used for genomic DNA
Insertion vectors: short vector with DNA of max 10kb
Used for cDNA
Cosmid vectors: hybrid between plasmid and phages by cloning a cos sequence in a plasmid
1. MboI cuts plasmid , BamHI cuts insert to create the same overhanging ends
2. Insert (max 30-45kb) ligated in the plasmid (use in vitro packaging mix)
3. Vector put in phase head using the cos sites
Combines plasmid convenience with phage efficiency
Fosmid: cosmid based on the F plasmid of E. Coli (lower copy number ORI – less DNA but more stable)
