Lecture 1 – What has biology ever done for us? (chapter 1 & 3)
Students are expected to be able to:
• Give examples of key differences between chemical and biological therapeutics
• Discuss the role of biotechnology in our modern pharmacopoeia
• Choose suitable restriction endonucleases for cloning a DNA fragment
• Analyze a Sanger sequencing trace
DNA basic tools
DNA representation
DNA always written in the 5’->3’ direction
The reverse complement 3’->5’ forms the double helix with the first strand
DNA-polymerase binds 3’-end of one of the DNA-strands and extends it in de direction of the
5’-end -> so the new DNA-strand is formed in the 5’->3’ direction
Strand = sense, plus, non-template or antisense, minus, template
o Non-template because protein synthesis in 5’->3’ (direction of polymerase, …)
2 types of plasmid DNA representation:
“Cut and paste” molecular biology
Nucleases -> cutting
Restriction endonucleases cut DNA at specific sequences (= restriction site)
Cut is not straight = sticky ends (cohesive ends) => easy to connect with other DNA-molecules
that are cut with the same restriction enzyme
➔ Cut could be straight = blunt ends
When methylase coupled to restriction endonuclease activity -> methylation of the restriction
enzyme in the host-DNA => no cutting (overlap restriction-site and methylation-site)
1-What has biology ever done for us? 1
,Orthodox Type II endonucleases
o Are dimers
o Recognize palindromic sequences of 4-8 bp
o Bv. 5’ – GAATTC – 3’
3’ – CTTAAG – 5’
• Palindromic because in the same reading direction 5’ -> 3’ both strands give
GAATTC
o Cut within the sequence site
o Example EcoRI
Ligases -> pasting
Enzymes that join DNA-ends and connect (ligates) 2 compatible ends with each other
It uses energy to ligate the DNA-ends
1. Enzyme binds ATP
2. Enzyme takes and binds AMP from ATP -> activation enzyme
3. Activated enzyme transfers AMP to the 5’-phosphate end of the DNA-strand -> activation
5’-phospate end
4. Activated 5’-phospate end attacks free 3’-OH in active centrum of enzyme
5. Phosphodiester bond formation -> no more cut in DNA, just one uninterrupted DNA-
strand
Sensitive to P-positioning
o The angle of the terminal phosphate is crucial for the working the ligase
• Helix-distortions that place the donor’s phosphate or acceptor’s hydroxyl out of the
right angle, interfere with the catalysis and so the ligation stops and the molecules are
not ligated
Example: EcoRI – G’ AATT, C
Exam-level exercise
1-What has biology ever done for us? 2
, Answer:
Agarose gel electrophoresis
Voltage across the gel => DNA separation by charge, in practice by length -> DNA visualization by
intercalating dye -> amplification of the correct fragment
First column = ladder -> size-reference
Restriction-analysis of DNA
Restriction-analysis of DNA:
1. Cutting DNA with restriction enzymes (endonucleases)
2. Separating DNA-fragments with agarose gel-electrophoresis
3. Visualizing DNA-fragments
4. Interpreting the results
➔ ‘+1’ is a reference point on the plasmid from where you can start counting to the cut-sites
Exam-level exercise
Answer:
1-What has biology ever done for us? 3
, DNA-assembly: biobricks
Classic molecular biology
Transferring a fragment from a source vector to a destination vector by using nucleases and
ligases to restrict and ligate DNA-parts
You have to comprise in some aspects
o No control over how close the fragments go -> creating gaps
o Can be an issue, because some elements are sensitive to distance
o No control over the fragment order
o Can be an issue, because the order of the fragments matter in the end-construct
Case-by-case answers and results
Expensive because you have to rely on multiplying the endonucleases
Example:
➔ You can’t cut H-P because in your destination vector you will create 3 fragments H-P, P-H
and H-H, which gives trouble and potential cloning problems
➔ Solution is to cut X-P in both source vector and destination vector because X-P doesn’t
have a compatible overhang and therefore can only ligate to their original site
o Red = isolated fragment from the source vector
o Green = backbone from the destination vector
➔ Ligating the isolated fragment into backbone
Important! A plasmid always needs an ‘origin of replication (ori)’ and an ‘antibiotic resistance
marker’ to function properly
o Ori = specific DNA-sequence that allows the plasmid to replicate
o Most common ‘colE1’
o Inserted gene or DNA of interest will not be lost when the host cell divides
o Antibiotic resistance marker = specific DNA-sequence that allows the host bacterial cell
to survive in the presence of a specific antibiotic
o Helps with distinguishing between cells with and without the plasmid so there
can be a selection that ensures only plasmid-bearing cells are cultured
Biobrick assembly
Biobricks = standardized DNA-fragments
Standard restriction enzymes = orthodox Type II endonucleases
o EcoRI
o XbaI
o SpeI
o PstI
• Recognize and cut specific sequences
1-What has biology ever done for us? 4
Students are expected to be able to:
• Give examples of key differences between chemical and biological therapeutics
• Discuss the role of biotechnology in our modern pharmacopoeia
• Choose suitable restriction endonucleases for cloning a DNA fragment
• Analyze a Sanger sequencing trace
DNA basic tools
DNA representation
DNA always written in the 5’->3’ direction
The reverse complement 3’->5’ forms the double helix with the first strand
DNA-polymerase binds 3’-end of one of the DNA-strands and extends it in de direction of the
5’-end -> so the new DNA-strand is formed in the 5’->3’ direction
Strand = sense, plus, non-template or antisense, minus, template
o Non-template because protein synthesis in 5’->3’ (direction of polymerase, …)
2 types of plasmid DNA representation:
“Cut and paste” molecular biology
Nucleases -> cutting
Restriction endonucleases cut DNA at specific sequences (= restriction site)
Cut is not straight = sticky ends (cohesive ends) => easy to connect with other DNA-molecules
that are cut with the same restriction enzyme
➔ Cut could be straight = blunt ends
When methylase coupled to restriction endonuclease activity -> methylation of the restriction
enzyme in the host-DNA => no cutting (overlap restriction-site and methylation-site)
1-What has biology ever done for us? 1
,Orthodox Type II endonucleases
o Are dimers
o Recognize palindromic sequences of 4-8 bp
o Bv. 5’ – GAATTC – 3’
3’ – CTTAAG – 5’
• Palindromic because in the same reading direction 5’ -> 3’ both strands give
GAATTC
o Cut within the sequence site
o Example EcoRI
Ligases -> pasting
Enzymes that join DNA-ends and connect (ligates) 2 compatible ends with each other
It uses energy to ligate the DNA-ends
1. Enzyme binds ATP
2. Enzyme takes and binds AMP from ATP -> activation enzyme
3. Activated enzyme transfers AMP to the 5’-phosphate end of the DNA-strand -> activation
5’-phospate end
4. Activated 5’-phospate end attacks free 3’-OH in active centrum of enzyme
5. Phosphodiester bond formation -> no more cut in DNA, just one uninterrupted DNA-
strand
Sensitive to P-positioning
o The angle of the terminal phosphate is crucial for the working the ligase
• Helix-distortions that place the donor’s phosphate or acceptor’s hydroxyl out of the
right angle, interfere with the catalysis and so the ligation stops and the molecules are
not ligated
Example: EcoRI – G’ AATT, C
Exam-level exercise
1-What has biology ever done for us? 2
, Answer:
Agarose gel electrophoresis
Voltage across the gel => DNA separation by charge, in practice by length -> DNA visualization by
intercalating dye -> amplification of the correct fragment
First column = ladder -> size-reference
Restriction-analysis of DNA
Restriction-analysis of DNA:
1. Cutting DNA with restriction enzymes (endonucleases)
2. Separating DNA-fragments with agarose gel-electrophoresis
3. Visualizing DNA-fragments
4. Interpreting the results
➔ ‘+1’ is a reference point on the plasmid from where you can start counting to the cut-sites
Exam-level exercise
Answer:
1-What has biology ever done for us? 3
, DNA-assembly: biobricks
Classic molecular biology
Transferring a fragment from a source vector to a destination vector by using nucleases and
ligases to restrict and ligate DNA-parts
You have to comprise in some aspects
o No control over how close the fragments go -> creating gaps
o Can be an issue, because some elements are sensitive to distance
o No control over the fragment order
o Can be an issue, because the order of the fragments matter in the end-construct
Case-by-case answers and results
Expensive because you have to rely on multiplying the endonucleases
Example:
➔ You can’t cut H-P because in your destination vector you will create 3 fragments H-P, P-H
and H-H, which gives trouble and potential cloning problems
➔ Solution is to cut X-P in both source vector and destination vector because X-P doesn’t
have a compatible overhang and therefore can only ligate to their original site
o Red = isolated fragment from the source vector
o Green = backbone from the destination vector
➔ Ligating the isolated fragment into backbone
Important! A plasmid always needs an ‘origin of replication (ori)’ and an ‘antibiotic resistance
marker’ to function properly
o Ori = specific DNA-sequence that allows the plasmid to replicate
o Most common ‘colE1’
o Inserted gene or DNA of interest will not be lost when the host cell divides
o Antibiotic resistance marker = specific DNA-sequence that allows the host bacterial cell
to survive in the presence of a specific antibiotic
o Helps with distinguishing between cells with and without the plasmid so there
can be a selection that ensures only plasmid-bearing cells are cultured
Biobrick assembly
Biobricks = standardized DNA-fragments
Standard restriction enzymes = orthodox Type II endonucleases
o EcoRI
o XbaI
o SpeI
o PstI
• Recognize and cut specific sequences
1-What has biology ever done for us? 4
