Molecular Biology: DNA Structure and Functions

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