Molecular Biology Flashcards
Pyrimidine bases
C,T,U
Nucleosides are
Base + sugar
Nucleotides are
Nucleoside + phosphate
DNA is polymerized in the _ to _ direction
5’ to 3’
Phosphodiester linkage
5’ PO4- to 3’ OH
3 types of DNA
A,B and Z
Differences in types of DNA
- Handedness: A-R, B-R, Z-L
- Condition: A-rel. humidity, B- high humidity, C-high salt
- BP/turn: A-11, B-10, C-12
- Vertical rise -B
- Helical Diameter -A
Replication is:
- Semi-conservative
2. Bidirectional (2 replication forks)
Mechanism of replication:
- Starts at origin of replication
- Local separation of duplex DNA
- Helicase unwinds the DNA at the fork
- SSB proteins keep duplex strands apart
- Primase synthesizes a short RNA primer
- DNA polymerase 3 extends this
- Topoisomerases maintain proper helical density
- Lagging strand synthesis involves Okazaki fragments
- DNA polymerase 1 removes RNA primers and fills the gaps
- Ligase seals the gaps
DNA polymerases can’t
- Melt or unwind duplex DNA
2. Initiate chains, can only extend a pre-existing DNA or RNA strand
Type 1 topoisomerases
Relax DNA by nicking and closing one strand of duplex DNA
Type 2 topoisomerases
Change DNA topology by breaking and rejoining double stranded DNA
Why is DNA replication accurate?
- Base pairing
- Proofreading by DNA polymerase
- Post-polymerase repair systems
How does DNA polymerase proofread?
- Senses the distortion of the double helix from insertion of incorrect bases
- Closes ‘fingers’ of hand
- Move DNA from polymerase domain to exonuclease domain where incorrect base is removed
- Opens fingers of hand
- DNA strand moves back to polymerase domain
Mismatch repair
Before replication finishes
Excision repair
After replication finishes
DNA polymerization reactions are performed in the presence of
Dideoxynucleotides
3 Main steps of PCR
- Denaturation
- Annealing
- Extension
Outline the Sanger method
- Nucleophilic attack by hydroxyl group hence di-deoxy
- Optimized conc. so that every time a given nucleotide is present + dideoxy incorporated sequencing is ended
- Nucleotide lengths separated by gel electrophoresis
- Different barcode pattern for each nucleotide so base sequence can be read by fragment length
Next generation sequencing…
Incorporates Sanger method but is much faster
