GENOME TECHNOLOGY AND
APPLICATIONS
CONTENT TABLE
Introduction ................................................................................................................................. 3
Cell based DNA cloning ................................................................................................................ 4
Principles of DNA cloning................................................................................................................. 4
Recombinant screening ..................................................................................................................12
Transformation ...............................................................................................................................14
Vectors ..........................................................................................................................................15
Classical site-directed mutagenesis ................................................................................................24
Expression cloning ...................................................................................................................... 26
Expression cloning in bacteria .........................................................................................................26
Cloning in eukaryotes .....................................................................................................................28
Expression in insect cells ................................................................................................................30
Semi stable expression cloning using SV40 ......................................................................................30
Expression cloning using viral vectors ..............................................................................................31
Stable expression in mammalian cells .............................................................................................32
PCR ............................................................................................................................................ 35
Self study: recent and convenient cloning systems ....................................................................... 47
1: Gateway system ..................................................................................................................... 47
2: Gibson assembly .................................................................................................................... 50
Exercise..................................................................................................................................... 51
Self study: recombinase polymerase amplification (RPA) .............................................................. 54
Model organisms, comparative genomics and evolution ............................................................... 58
Model organisms ............................................................................................................................58
Unicellular organisms ................................................................................................................ 58
Invertebrates ............................................................................................................................. 59
Vertebrates................................................................................................................................ 61
Comparative genomics ...................................................................................................................62
Evolution........................................................................................................................................65
Pharmacogenetics and genomics ................................................................................................ 73
1
,Genome technologies seminar: feedback class ............................................................................ 86
Multiplex families (different inheritance patterns) .............................................................................86
Isolated patients with (rare) unresolved presumed genetic disorders ................................................87
Example 1: Miller Syndrome (recessive) ...................................................................................... 87
Example 2: Helsmoortel – van der Aa Syndrome (de novo) ............................................................ 89
Screening large cohorts (additional evidence) & diagnostic screening ...............................................91
What about genetic disorders caused by repeat expansions, structural variation…? ..........................93
Can balanced events be identified? .................................................................................................93
And copy number variations (CNVs)?...............................................................................................94
Epigenetics and disease .................................................................................................................96
De novo assembly of ‘novel’ genomes (biology)................................................................................97
Disease modelling – understanding the pathomechanics .................................................................97
Genome editing: CRISPR-Cas..................................................................................................... 100
Genome editing ............................................................................................................................ 100
CRISPR-Cas ................................................................................................................................. 103
Exerises session........................................................................................................................ 109
Induced pluripotent stem cells (iPSCs) ....................................................................................... 115
Definitions and general principles ................................................................................................. 115
Experimental approach................................................................................................................. 121
In the lab ...................................................................................................................................... 128
Current limitations ....................................................................................................................... 129
2
,INTRODUCTION
• Exam
o Written, in English
o About 6 open questions, at least 1 from each teacher
o Different sheet of paper for each question
3
,CELL BASED DNA CLONING
PRINCIPLES OF DNA CLONING
• Piece of human DNA and piece of bacterial DNA and put it together
• Cell based DNA cloning comprises 4 steps:
o 1: In vitro construction of a recombinant DNA molecule
§ Cut pieces of different DNA and put them together
o 2: Transformation
§ Transform recombinant DNA in a host (often E. coli, can be a yeast
too)
o 3: Selective propagation of clones
§ Make sure it doesn’t go lost, it has to be replicated
o 4: Isolation of recombinant DNA clones
§ Lysis of host
• 1: In vitro construction of a recombinant DNA molecule
o Requires cutting and pasting of DNA
§ Restriction endonucleases: cut DNA molecule)
§ DNA ligase (glue): put it back together
o Requires a replicon
§ A piece of DNA that makes independent DNA replication possible
§ Replicon is specific for a host
§ Usually a construct called “vector” is used, containing many
features used in the cloning process
o
§ Bacterial cells that contain extrachromosomal replicon
§ Plasmids are often used as a vector
§ Chromosome is present in 1 copy
§ Plasmids are present in many copies and replicate independently
§ Cut with restriction enzyme, ligation à recombinant DNA
4
,• 2: Transformation
o Recombinant DNA molecule is introduced in a host cell
o Usually bacterium or yeast
§ Easy to grow
§ Fast reproduction
o For expression studies, cloning is often done in eukaryotic cells
(mammalian cells, insect cells, see later in this chapter)
§ But a lot harder
§ But we have to use eukaryotic cells sometimes because E. coli
cannot synthetize big (human) proteins
o For expression studies, cloning in a bacterium usually precedes cloning in
the host used for the expression
§ All the cutting and pasting in E. coli but no expression yet, this is
fine even with big proteins
§ So you always need E. coli, at least for the preparation
• 3: Selective propagation of clones
o Cells are plated on agar
o Each individual cell forms a colony
o Each colony is a clone:
All cells are identical, and have the same ancestor cell
o 1 colony can be grown in liquid medium to obtain more cells
§ Left: impossible to count (you need to dilute it)
5
,• 4: Isolation of recombinant DNA clones
o The recombinant DNA is purified from the cells
§ Take out recombinant DNA
o This you can use for many experiments
• Restriction endonucleases
o Nomenclature: 1 letter genus, 2 letters species, followed by number
E.g. HaeIII: Hemophilus aegypticus
o Defence mechanism against bacteriophages
§ DNA from bacteriophage enters the bacterium and is cleaved
§ Cleaved: then virus is destroyed and in won’t affect the bacterium
§ There is a matching sequence specific DNA methylase
> Methylation of the same recognition sequence à not
recognized by RE
> Bacterial DNA cannot be cut
§ Type II RE will cut a specific recognition sequence
§ Usually 4-8 bp
§ Usually palindrome (same from left to right as from right to left)
o Cleavage
§ On the symmetry axis: blunt ends (when you cut straight in the
middle of the palindrome: see green line in figure)
§ Usually not on the symmetry axis: overhangs = sticky ends =
cohesive termini (see figure) (when you don’t cut straight)
> 3 prime end: DNA polymerase will do its work and extend
the molecule
> Sequence always notated from 5 prime end to 3 prime end
6
,§
> Left: palindrome of the complementary strands
> Left: arrows are where we cut: 2 cuts close together à
molecule will fall apart
> If 2 cuts are far apart (more than 10-15 bp) the molecule
doesn’t fall apart because there are many hydrogen bonds
in between
§ Sticky ends can base pair and form unstable double helices
> 5’ overhang
> 3’ overhang
7
,o Restriction enzymes
o
§ Expected size after cutting (some a little longer or shorter)
§ Less GC (40%) than AT in human genome
§ CpG site: site where methylation occurs in genome (epigenetics:
determines which genes in cells are expressed)
> Inherited in cell lineages, liver cell has an epigenetic imprint
for example à 2 daughter cells will also be liver cells
> Only cuts 1/5th of expected frequency
> Longer size with CG and CpG
o Isoschizomeres: different restriction enzymes that have the same
recognition sequence
§ They cut the same enzymes
o Some restriction endonucleases (ER) have compatible cohesive termini
E.g. BamHI: G¯GATCC en MboI: ¯GATC
8
, o Examples of commonly used ER
• DNA ligase
o DNA ligase can restore a
covalent bond in a DNA
molecule
o Sticky ends that are hybridized
together are instable (because
it’s just a couple of bases),
even at low temperature
o The hydrogen bonds of the
double helix facilitate the
work of DNA ligase
§ It’s easier to ligate
sticky ands than blunt
ends
o Several different fragments
that are ligated together are
called a concatamere
§ You might ligate other
pieces of DNA together à you might ligate everything together in
really long pieces
o Intramolecular ligation = (vector) cyclisation
§ Without insert in vector
o You might close the vector when the 2 ends of the vector are close
together
9
, • Origin of replication (ori)
o An ORI allows replication independent of the host chromosome
o Independent replication facilitates purification of recombinant molecule
o Bacterial chromosome:
§ Circular chromosome with 1 ORI
• Human chromosome has a few, otherwise it would take
very long to replicate
§ ORI limits the number of chromosomes to 1 per cell
• Mosed used vectors
o Plasmids
§ Small circular DNA molecules in bacteria
§ Usually multiple copy number ORI
§ Found in nature
§ Usually contain only a few genes
§ Transmitted vertically from parental to daughter cells
§ Transmitted horizontally from one bacterium to another via
conjugation
> This is a problem in
hospitals with
antibiotical resistance
§ Have a supercoil structure
(same as bacterial
chromosome)
> A helix is twisted
> Supercoil is an
additional twist à
circular DNA-molecule
will coil up
> Replication causes
supercoil because the
DNA get extra twists
> We can use the
supercoil to purify the
plasmids (see further)
o Bacteriophages
§ Viruses that infect bacteria
§ Extremely efficient transformation
§ Linear or circular genome
§ Can be found outside the cell in a protein coat
10
APPLICATIONS
CONTENT TABLE
Introduction ................................................................................................................................. 3
Cell based DNA cloning ................................................................................................................ 4
Principles of DNA cloning................................................................................................................. 4
Recombinant screening ..................................................................................................................12
Transformation ...............................................................................................................................14
Vectors ..........................................................................................................................................15
Classical site-directed mutagenesis ................................................................................................24
Expression cloning ...................................................................................................................... 26
Expression cloning in bacteria .........................................................................................................26
Cloning in eukaryotes .....................................................................................................................28
Expression in insect cells ................................................................................................................30
Semi stable expression cloning using SV40 ......................................................................................30
Expression cloning using viral vectors ..............................................................................................31
Stable expression in mammalian cells .............................................................................................32
PCR ............................................................................................................................................ 35
Self study: recent and convenient cloning systems ....................................................................... 47
1: Gateway system ..................................................................................................................... 47
2: Gibson assembly .................................................................................................................... 50
Exercise..................................................................................................................................... 51
Self study: recombinase polymerase amplification (RPA) .............................................................. 54
Model organisms, comparative genomics and evolution ............................................................... 58
Model organisms ............................................................................................................................58
Unicellular organisms ................................................................................................................ 58
Invertebrates ............................................................................................................................. 59
Vertebrates................................................................................................................................ 61
Comparative genomics ...................................................................................................................62
Evolution........................................................................................................................................65
Pharmacogenetics and genomics ................................................................................................ 73
1
,Genome technologies seminar: feedback class ............................................................................ 86
Multiplex families (different inheritance patterns) .............................................................................86
Isolated patients with (rare) unresolved presumed genetic disorders ................................................87
Example 1: Miller Syndrome (recessive) ...................................................................................... 87
Example 2: Helsmoortel – van der Aa Syndrome (de novo) ............................................................ 89
Screening large cohorts (additional evidence) & diagnostic screening ...............................................91
What about genetic disorders caused by repeat expansions, structural variation…? ..........................93
Can balanced events be identified? .................................................................................................93
And copy number variations (CNVs)?...............................................................................................94
Epigenetics and disease .................................................................................................................96
De novo assembly of ‘novel’ genomes (biology)................................................................................97
Disease modelling – understanding the pathomechanics .................................................................97
Genome editing: CRISPR-Cas..................................................................................................... 100
Genome editing ............................................................................................................................ 100
CRISPR-Cas ................................................................................................................................. 103
Exerises session........................................................................................................................ 109
Induced pluripotent stem cells (iPSCs) ....................................................................................... 115
Definitions and general principles ................................................................................................. 115
Experimental approach................................................................................................................. 121
In the lab ...................................................................................................................................... 128
Current limitations ....................................................................................................................... 129
2
,INTRODUCTION
• Exam
o Written, in English
o About 6 open questions, at least 1 from each teacher
o Different sheet of paper for each question
3
,CELL BASED DNA CLONING
PRINCIPLES OF DNA CLONING
• Piece of human DNA and piece of bacterial DNA and put it together
• Cell based DNA cloning comprises 4 steps:
o 1: In vitro construction of a recombinant DNA molecule
§ Cut pieces of different DNA and put them together
o 2: Transformation
§ Transform recombinant DNA in a host (often E. coli, can be a yeast
too)
o 3: Selective propagation of clones
§ Make sure it doesn’t go lost, it has to be replicated
o 4: Isolation of recombinant DNA clones
§ Lysis of host
• 1: In vitro construction of a recombinant DNA molecule
o Requires cutting and pasting of DNA
§ Restriction endonucleases: cut DNA molecule)
§ DNA ligase (glue): put it back together
o Requires a replicon
§ A piece of DNA that makes independent DNA replication possible
§ Replicon is specific for a host
§ Usually a construct called “vector” is used, containing many
features used in the cloning process
o
§ Bacterial cells that contain extrachromosomal replicon
§ Plasmids are often used as a vector
§ Chromosome is present in 1 copy
§ Plasmids are present in many copies and replicate independently
§ Cut with restriction enzyme, ligation à recombinant DNA
4
,• 2: Transformation
o Recombinant DNA molecule is introduced in a host cell
o Usually bacterium or yeast
§ Easy to grow
§ Fast reproduction
o For expression studies, cloning is often done in eukaryotic cells
(mammalian cells, insect cells, see later in this chapter)
§ But a lot harder
§ But we have to use eukaryotic cells sometimes because E. coli
cannot synthetize big (human) proteins
o For expression studies, cloning in a bacterium usually precedes cloning in
the host used for the expression
§ All the cutting and pasting in E. coli but no expression yet, this is
fine even with big proteins
§ So you always need E. coli, at least for the preparation
• 3: Selective propagation of clones
o Cells are plated on agar
o Each individual cell forms a colony
o Each colony is a clone:
All cells are identical, and have the same ancestor cell
o 1 colony can be grown in liquid medium to obtain more cells
§ Left: impossible to count (you need to dilute it)
5
,• 4: Isolation of recombinant DNA clones
o The recombinant DNA is purified from the cells
§ Take out recombinant DNA
o This you can use for many experiments
• Restriction endonucleases
o Nomenclature: 1 letter genus, 2 letters species, followed by number
E.g. HaeIII: Hemophilus aegypticus
o Defence mechanism against bacteriophages
§ DNA from bacteriophage enters the bacterium and is cleaved
§ Cleaved: then virus is destroyed and in won’t affect the bacterium
§ There is a matching sequence specific DNA methylase
> Methylation of the same recognition sequence à not
recognized by RE
> Bacterial DNA cannot be cut
§ Type II RE will cut a specific recognition sequence
§ Usually 4-8 bp
§ Usually palindrome (same from left to right as from right to left)
o Cleavage
§ On the symmetry axis: blunt ends (when you cut straight in the
middle of the palindrome: see green line in figure)
§ Usually not on the symmetry axis: overhangs = sticky ends =
cohesive termini (see figure) (when you don’t cut straight)
> 3 prime end: DNA polymerase will do its work and extend
the molecule
> Sequence always notated from 5 prime end to 3 prime end
6
,§
> Left: palindrome of the complementary strands
> Left: arrows are where we cut: 2 cuts close together à
molecule will fall apart
> If 2 cuts are far apart (more than 10-15 bp) the molecule
doesn’t fall apart because there are many hydrogen bonds
in between
§ Sticky ends can base pair and form unstable double helices
> 5’ overhang
> 3’ overhang
7
,o Restriction enzymes
o
§ Expected size after cutting (some a little longer or shorter)
§ Less GC (40%) than AT in human genome
§ CpG site: site where methylation occurs in genome (epigenetics:
determines which genes in cells are expressed)
> Inherited in cell lineages, liver cell has an epigenetic imprint
for example à 2 daughter cells will also be liver cells
> Only cuts 1/5th of expected frequency
> Longer size with CG and CpG
o Isoschizomeres: different restriction enzymes that have the same
recognition sequence
§ They cut the same enzymes
o Some restriction endonucleases (ER) have compatible cohesive termini
E.g. BamHI: G¯GATCC en MboI: ¯GATC
8
, o Examples of commonly used ER
• DNA ligase
o DNA ligase can restore a
covalent bond in a DNA
molecule
o Sticky ends that are hybridized
together are instable (because
it’s just a couple of bases),
even at low temperature
o The hydrogen bonds of the
double helix facilitate the
work of DNA ligase
§ It’s easier to ligate
sticky ands than blunt
ends
o Several different fragments
that are ligated together are
called a concatamere
§ You might ligate other
pieces of DNA together à you might ligate everything together in
really long pieces
o Intramolecular ligation = (vector) cyclisation
§ Without insert in vector
o You might close the vector when the 2 ends of the vector are close
together
9
, • Origin of replication (ori)
o An ORI allows replication independent of the host chromosome
o Independent replication facilitates purification of recombinant molecule
o Bacterial chromosome:
§ Circular chromosome with 1 ORI
• Human chromosome has a few, otherwise it would take
very long to replicate
§ ORI limits the number of chromosomes to 1 per cell
• Mosed used vectors
o Plasmids
§ Small circular DNA molecules in bacteria
§ Usually multiple copy number ORI
§ Found in nature
§ Usually contain only a few genes
§ Transmitted vertically from parental to daughter cells
§ Transmitted horizontally from one bacterium to another via
conjugation
> This is a problem in
hospitals with
antibiotical resistance
§ Have a supercoil structure
(same as bacterial
chromosome)
> A helix is twisted
> Supercoil is an
additional twist à
circular DNA-molecule
will coil up
> Replication causes
supercoil because the
DNA get extra twists
> We can use the
supercoil to purify the
plasmids (see further)
o Bacteriophages
§ Viruses that infect bacteria
§ Extremely efficient transformation
§ Linear or circular genome
§ Can be found outside the cell in a protein coat
10
