Deze samenvatting bevat alle onderwerpen behandeld tijdens minor 13 (molecular cloning, genetically modified organisms, CRISPR/Cas9, vaccines, therapeutic antibodies, genetic therapy and its tool box, molecular diagnostics, gene therapy, stem cells & tissue engineering en research involving humans).
Index
Molecular cloning..............................................................................................................................4
DNA cloning in the former century.......................................................................................................................4
Analyzing and modifying DNA...............................................................................................................................6
Sanger sequencing..........................................................................................................................................6
Next generation sequencing...........................................................................................................................7
Transferring DNA into the host.............................................................................................................................7
CRISPR/Cas technologies.................................................................................................................11
Prime editing.......................................................................................................................................................12
Genetic therapy and its Tool box.....................................................................................................15
Biotechnological toolbox and corresponding strategies.....................................................................................15
Duchenne Muscular Dystrophy...........................................................................................................................16
Spinal Muscular Atrophy (SMA)..........................................................................................................................16
Cystic fibrosis.......................................................................................................................................................17
Molecular diagnostics......................................................................................................................17
Next generation sequencing via single molecule Molecular Inversion Probes (ssMIP).....................................17
Concepts of NGS............................................................................................................................................18
Copy number variations (CNV) detection by NGS.........................................................................................18
Biomaterials for the scaffolds.......................................................................................................................21
Research involving humans: legal and ethical aspects.....................................................................23
Biobank................................................................................................................................................................23
Medical Research Involving Human Subjects Act (Wet Mensgebonden Onderzoek, WMO).............................23
3
, Medical Biotechnology towards Clinical Practice
Molecular cloning
DNA cloning in the former century
Exonucleases: enzymes that cleave nucleotides off one by one from the end (exo) of a
polynucleotide chain.
Endonucleases: enzymes that cleave phosphodiester bonds in the middle (endo) of a
polynucleotide chain.
Restriction enzymes: are endonucleases that cut double-stranded DNA at sequence-
specific sites (restriction sites) in a very defined way.
EcoRI (restriction enzyme I from E. coli), for example, recognizes the sequence "GAATTC" and
produces "sticky" ends (that read AATT). This creates a 5’ overhanging end. These sticky ends
can be (re)connected by ligase via phosphodiester bond formation. Importantly, there are two
requirements that need to be fulfilled for ligase to be able to do its job:
1. Only fragments a' and a fit together. The ends in b and c do not match with a'
(remember the base pair rules).
2. Ligase needs a 5'-phosphate group to be present to connect it to the 3'-OH group of the
other fragment.
In the case that blunt ends are needed for cloning purposes, DNA fragments with 5' overhanging
ends can be 'filled-up' using Klenow fragment (a special DNA polymerase part). The only option
to blunt 3’ overhanging sites would be to eat the overhang away using a nuclease that displays 3'
exonuclease activity. All DNA polymerases with proofreading capability know this trick.
Plasmids are small, circular DNA molecules that are not part of the chromosomal DNA of
bacteria. They replicate independently, exploiting the host machinery, and confer selective
advantage (e.g. antibiotic resistance).
↪ Artificial plasmids are widely used as vectors in molecular cloning and are made non-
conjugating to prevent horizontal gene transfer.
Origin of replication: DNA sequence which allows initiation of replication
within a plasmid by recruiting replication machinery proteins.
Antibiotic resistance gene: Allows for selection of plasmid-containing
bacteria.
Multiple Cloning Site (MCS): Short segment of DNA which contains several
restriction sites allowing for the easy insertion of DNA. In expression
plasmids, the MCS is often downstream from a promoter.
Insert: Gene, promoter or other DNA fragment cloned into the MCS for
further study.
Promotor region: Drives transcription of the target gene. Vital component
for expression vectors: determines which cell types the gene is expressed in
and amount of recombinant protein obtained.
Selectable marker: Many plasmids also have selectable markers for use in
other cell types.
1. The first step in cloning a gene is to isolate the DNA from the
organism that contains the desired gene.
2. The isolated DNA is purified and then fragmented with a restriction enzyme.
Each fragment has a single-stranded sequence of nucleotides on its ends that is
capable of hybridizing with DNA that has been fragmented with the same
restriction enzyme.
3. The DNA fragments are then incorporated into plasmids. The type of plasmid
used for cloning has a single restriction site, and when cleaved by the
restriction enzyme, generates the same cohesive ends that are in the fragments of the DNA to be
cloned.
4
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