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
Exam questions ......................................................................................................................... 131
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
▪ 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 → 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 Defense 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 → 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 →
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
▪ 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 → 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: GGATCC 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 concatemer
▪ You might ligate other
pieces of DNA together → you might ligate everything together in
really long pieces
o Intramolecular ligation = (vector) cyclization
▪ 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
• Most 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 →
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
Exam questions ......................................................................................................................... 131
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
▪ 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 → 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 Defense 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 → 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 →
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
▪ 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 → 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: GGATCC 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 concatemer
▪ You might ligate other
pieces of DNA together → you might ligate everything together in
really long pieces
o Intramolecular ligation = (vector) cyclization
▪ 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
• Most 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 →
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
