Podcast
Questions and Answers
What is the term for the process of reannealing of DNA?
What is the term for the process of reannealing of DNA?
What is the wavelength of UV light that DNA and RNA absorb due to aromatic bases?
What is the wavelength of UV light that DNA and RNA absorb due to aromatic bases?
What is the effect of base stacking on the absorbance of DNA?
What is the effect of base stacking on the absorbance of DNA?
What is the primary function of restriction-modification systems in bacteria?
What is the primary function of restriction-modification systems in bacteria?
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What is the type of restriction enzymes that recognise palindromic sequences?
What is the type of restriction enzymes that recognise palindromic sequences?
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What is the maximum size of DNA fragments that can be carried by plasmids and bacteriophages?
What is the maximum size of DNA fragments that can be carried by plasmids and bacteriophages?
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What is the purpose of using ethidium bromide in DNA analysis?
What is the purpose of using ethidium bromide in DNA analysis?
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What is the purpose of the CRISPR system in microbes?
What is the purpose of the CRISPR system in microbes?
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How do DNA endonucleases distinguish between bacterial and viral DNA?
How do DNA endonucleases distinguish between bacterial and viral DNA?
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What is the purpose of methylating DNA in cells?
What is the purpose of methylating DNA in cells?
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What is the characteristic of Type II restriction enzymes?
What is the characteristic of Type II restriction enzymes?
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What is the purpose of using bacteriophages in DNA cloning?
What is the purpose of using bacteriophages in DNA cloning?
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What is the limitation of using plasmids and bacteriophages for DNA cloning?
What is the limitation of using plasmids and bacteriophages for DNA cloning?
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What is the purpose of the short viral DNA sequences in the microbial genome?
What is the purpose of the short viral DNA sequences in the microbial genome?
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What is the function of CAS9 nuclease in the CRISPR system?
What is the function of CAS9 nuclease in the CRISPR system?
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What is the application of CRISPR/Cas9 in treating genetic disorders?
What is the application of CRISPR/Cas9 in treating genetic disorders?
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What is the outcome of the CRISPR/Cas9 system during infection?
What is the outcome of the CRISPR/Cas9 system during infection?
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What is the type of genetic disorders that CRISPR/Cas9 is approved to treat?
What is the type of genetic disorders that CRISPR/Cas9 is approved to treat?
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Study Notes
DNA Structure and Properties
- DNA absorbs UV light at 260nm due to aromatic bases
- Double helix has reduced absorbance vs denatured state due to base stacking = hyperchromicity effect
DNA Hybridisation and PCR
- DNA hybridisation = reannealing, involved in PCR, genotyping by in situ hybridisation, Southern blotting, DNA microarrays (chips)
- PCR uses Taq polymerase, active for over 40 cycles of heating and cooling
Restriction Endonucleases
- DNA endonucleases are bacterial enzymes that destroy incoming viral DNA = RESTRICTION-MODIFICATION SYSTEM
- Restriction endonucleases distinguish between bacterial and viral DNA by recognition of particular sequences absent from bacterial genome
- Type II restriction enzymes are most common, recognise PALINDROMIC SEQUENCES (few-tens bps)
- Can cut to generate two blunt ends OR sticky ends
Cloning Vectors
- Plasmids and bacteriophages can be used to carry DNA
- Restriction endonucleases and DNA ligase cut/paste DNA into phage genome, copied with virus in bacteria
- Longer fragments need yeast/bacteria artificial chromosomes, max for plasmids/bacteriophages = 1000s bp
DNA Separation and Visualisation
- Negatively charged DNA can be separated by size in electric field
- Visualised by dyes binding dsDNA and fluoresce under UV light, common dye = ETHIDIUM BROMIDE (mutagen)
Southern Blot
- Transfer from gel to filter, analyse by hybridisation to see if separated DNA contains target sequences
CRISPR-Cas9
- Based on immunity in Archaea and bacteria, protecting against viruses
- CRISPR = short viral DNA sequences in microbial genome which are transcribed to RNA, used as guides to direct CAS9 NUCLEASE to cleave DNA of invading virus during infection
- Approved for treatments for sickle-cell anaemia and beta-thalassemia, reactivating fetal haemoglobin in patients with defective adult haemoglobin
Restriction-Modification System
- Bacteria possess DNA endonucleases that destroy incoming viral DNA to restrict viral infection.
- Bacteria modify their own DNA to avoid self-destruction by these enzymes.
DNA Methylation
- DNA methylation occurs at adenine or cytosine residues in cells.
Type II Restriction Enzymes
- Most common type of restriction enzymes.
- Recognize palindromic sequences (few to tens of base pairs).
- Can generate either blunt ends or sticky ends when cutting DNA.
DNA Cloning
- Plasmids can be used to carry DNA.
- Bacteriophages (viruses that infect bacteria) can also be used to carry DNA.
- Restriction endonucleases and DNA ligase are used to cut and paste DNA into phage genome.
- The recombinant phage genome is then copied within bacteria.
- Yeast/bacteria artificial chromosomes are required for longer fragments (>1000s bp).
CRISPR/Cas9 System
- Derived from the immune system of Archaea and bacteria, which protects against viruses
- CRISPR contains short viral DNA sequences in the microbial genome
- These sequences are transcribed to RNA, which acts as a guide
Mechanism of Action
- The RNA guide directs the CAS9 nuclease to cleave the DNA of invading viruses during infection
- CAS9 nuclease cleaves the viral DNA, preventing viral replication
Medical Applications
- Approved for the treatment of sickle-cell anaemia
- Approved for the treatment of beta-thalassemia
- Reactivates fetal haemoglobin in patients with defective adult haemoglobin
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Description
This quiz covers the fundamentals of DNA structure, including DNA melting and hybridization, absorption of UV light, and the role of DNA endonucleases in bacterial defense systems.
