RECOMBINANT DNA TECHNOLOGY
The chemical structure of DNA includes a backbone made of alternating sugar
and phosphate. Attached to the sugar group is a base, either adenine, thymine,
guanine or cytosine (A-T and G-C). The two strands are connected through
hydrogen bonds between the base pairs. A-T has two hydrogen bridges, G-C has
three hydrogen bridges. DNA is transcribed to mRNA (with uracil instead of
thymine) by RNA polymerase, the mRNA is translated to amino-acid sequences,
forming proteins. DNA has an orientation. The 5’ side has a free hydroxyl or
phosphate on the 5’ carbon atom of the sugar ring and the 3’ side has a free
hydroxyl or phosphate on the 3’ carbon atom. Replication and transcription occur
in the 5’ to 3’ direction.
A gene is a segment of DNA that encodes the information for a certain trait and
is transcribed and translated. The exons of genes form the mRNA, the introns are
cut out during splicing. Thus, genes are templates for the production of RNA
and/or proteins. Proteins consist of a sequence of amino acids, there are 20
amino acids that are incorporated in proteins. ORFs, open reading frames, are
the parts of the DNA between the start and stop codon, which are transcribed. A
genome is the collective inheritable material of an organism and contains all
necessary information to encode all functional unites required for the
development of an organism. This includes chromosomal DNA, plastid DNA and
other genetic elements such as plasmids.
The Human Genome Project was launched in 1990, in 2003 the first complete
euchromatin part of the human genome sequence was finished. Euchromatin is
lightly packed DNA that often contains many genes and is under active
transcription. The other form is heterochromatin, which is tightly packed and less
accessible for transcription. In 2021 the full telomere to telomere sequence of
the human genome was mapped.
Functional genomics refers to the study of understanding the gene function and
interactions to be able to establish a relationship between an organism’s genome
and its phenotype. It gives an overall picture of genome functions, including the
expression profiles at the mRNA and protein levels.
There are multiple ways in which DNA changes can be introduced in vivo.
Random mutagenesis can be physical (UV, heavy ions, X-ray, gamma radiation,
atmospheric and room temperature plasma), biological/insertional (viruses,
transposons, bacteria) and chemical (5’-bromouracil, ethyl methane sulfonate,
methyl methane sulphonate, nitro-N-nitrosoguanidine). Non-ionizing radiation
does not lead to mutations, ionizing radiation (UV, X-rays, gamma rays) does.
This occurs because ionizing radiation causes double stranded breaks, which can
be incorrectly repaired, leading to mutations. EMS is an alkylating agent that
induces chemical modifications of nucleotides, resulting in base pair changes
during duplication. EMS mainly induces changes in guanine, causing G-C to
become A-T. 5-BU is a base analog of thymine that induces base pairing errors
during replication, because it can take two forms. It can be incorporated in the
place of thymine and eventually lead to T to C or G to A transition mutations.
When genes are discovered, based on the mutant phenotypes that occur after
random mutagenesis, this is called forward genetics. Reverse genetics works the
other way: a specific gene is mutated and its function is derived from the
phenotype that occurs.
, Targeted editing can occur through Cre-Lox recombination, transcription
activator-like effector nucleases (TALENs), CRISPR/Cas and zinc-finger nucleases
(crisprs). TALEN is an engineered product that is initially found in a plant-
pathogenic bacteria. It causes target specific double stranded breaks and can
result in various mutations, including deletions, inversions, insertions and
translocations. TALEN recognizes the target site based on DNA-protein
interaction, CRISPR is based on site specific RNA-protein interactions.
Target DNA or a gene of interest can be amplified using cloning. Before
designing a cloning construct, it is important to consider what the designated
bacterial cell line or strain is, what the selectable marker in the backbone of the
construct is, what antibiotic resistance genes the construct has, what promotor
and terminator can be used and what cloning method can be used. There are
many cloning methods: restriction enzymes and T4 ligase, PCR cloning, ligation-
independent cloning (no T4 ligase), Gibson seamless cloning, Gateway
recombination cloning, In-Fusion seamless cloning, SLICE cloning, Golden Gate
modular cloning. Gibson cloning starts with DNA fragments with overlapping
ends, this is added to the master mix and heated to 50 degrees, the exonuclease
chews back the 5’ ends, the fragments then anneal and the gaps are closed and
sealed by DNA polymerase and DNA ligase.
Gateway cloning involves two steps based on the integration and excision
reaction of the bacteriophage lambda. In-Fusion cloning works by fusing
overlapping ends of the fragments in a vector. Golden Gate cloning uses Type IIS
restriction enzymes and T4 DNA ligase. Vectors or plasmids can enter a cell
through heat shock or electric shock.
The chemical structure of DNA includes a backbone made of alternating sugar
and phosphate. Attached to the sugar group is a base, either adenine, thymine,
guanine or cytosine (A-T and G-C). The two strands are connected through
hydrogen bonds between the base pairs. A-T has two hydrogen bridges, G-C has
three hydrogen bridges. DNA is transcribed to mRNA (with uracil instead of
thymine) by RNA polymerase, the mRNA is translated to amino-acid sequences,
forming proteins. DNA has an orientation. The 5’ side has a free hydroxyl or
phosphate on the 5’ carbon atom of the sugar ring and the 3’ side has a free
hydroxyl or phosphate on the 3’ carbon atom. Replication and transcription occur
in the 5’ to 3’ direction.
A gene is a segment of DNA that encodes the information for a certain trait and
is transcribed and translated. The exons of genes form the mRNA, the introns are
cut out during splicing. Thus, genes are templates for the production of RNA
and/or proteins. Proteins consist of a sequence of amino acids, there are 20
amino acids that are incorporated in proteins. ORFs, open reading frames, are
the parts of the DNA between the start and stop codon, which are transcribed. A
genome is the collective inheritable material of an organism and contains all
necessary information to encode all functional unites required for the
development of an organism. This includes chromosomal DNA, plastid DNA and
other genetic elements such as plasmids.
The Human Genome Project was launched in 1990, in 2003 the first complete
euchromatin part of the human genome sequence was finished. Euchromatin is
lightly packed DNA that often contains many genes and is under active
transcription. The other form is heterochromatin, which is tightly packed and less
accessible for transcription. In 2021 the full telomere to telomere sequence of
the human genome was mapped.
Functional genomics refers to the study of understanding the gene function and
interactions to be able to establish a relationship between an organism’s genome
and its phenotype. It gives an overall picture of genome functions, including the
expression profiles at the mRNA and protein levels.
There are multiple ways in which DNA changes can be introduced in vivo.
Random mutagenesis can be physical (UV, heavy ions, X-ray, gamma radiation,
atmospheric and room temperature plasma), biological/insertional (viruses,
transposons, bacteria) and chemical (5’-bromouracil, ethyl methane sulfonate,
methyl methane sulphonate, nitro-N-nitrosoguanidine). Non-ionizing radiation
does not lead to mutations, ionizing radiation (UV, X-rays, gamma rays) does.
This occurs because ionizing radiation causes double stranded breaks, which can
be incorrectly repaired, leading to mutations. EMS is an alkylating agent that
induces chemical modifications of nucleotides, resulting in base pair changes
during duplication. EMS mainly induces changes in guanine, causing G-C to
become A-T. 5-BU is a base analog of thymine that induces base pairing errors
during replication, because it can take two forms. It can be incorporated in the
place of thymine and eventually lead to T to C or G to A transition mutations.
When genes are discovered, based on the mutant phenotypes that occur after
random mutagenesis, this is called forward genetics. Reverse genetics works the
other way: a specific gene is mutated and its function is derived from the
phenotype that occurs.
, Targeted editing can occur through Cre-Lox recombination, transcription
activator-like effector nucleases (TALENs), CRISPR/Cas and zinc-finger nucleases
(crisprs). TALEN is an engineered product that is initially found in a plant-
pathogenic bacteria. It causes target specific double stranded breaks and can
result in various mutations, including deletions, inversions, insertions and
translocations. TALEN recognizes the target site based on DNA-protein
interaction, CRISPR is based on site specific RNA-protein interactions.
Target DNA or a gene of interest can be amplified using cloning. Before
designing a cloning construct, it is important to consider what the designated
bacterial cell line or strain is, what the selectable marker in the backbone of the
construct is, what antibiotic resistance genes the construct has, what promotor
and terminator can be used and what cloning method can be used. There are
many cloning methods: restriction enzymes and T4 ligase, PCR cloning, ligation-
independent cloning (no T4 ligase), Gibson seamless cloning, Gateway
recombination cloning, In-Fusion seamless cloning, SLICE cloning, Golden Gate
modular cloning. Gibson cloning starts with DNA fragments with overlapping
ends, this is added to the master mix and heated to 50 degrees, the exonuclease
chews back the 5’ ends, the fragments then anneal and the gaps are closed and
sealed by DNA polymerase and DNA ligase.
Gateway cloning involves two steps based on the integration and excision
reaction of the bacteriophage lambda. In-Fusion cloning works by fusing
overlapping ends of the fragments in a vector. Golden Gate cloning uses Type IIS
restriction enzymes and T4 DNA ligase. Vectors or plasmids can enter a cell
through heat shock or electric shock.
