Lecture 2 Flashcards
what kind of replication
semi-conservative
double helix, template strand, synthesis 2 new strands
prokaryotic replication
single circular chromosome
bidirectional
single origin of replication (ori)
direction of replication
synthesis of DNA/RNA always in 5’ - 3’ direction
parental template run in 3’ to 5’ direction
helicase enzyme
unwinding/pulls apart helical double stranded DNA
gives 2 parental templates
Primase enzyme
starting point for nucleotide addition
short strand of RNA
DNA Polymerase 3 enzyme
Progressive addition of new nucleotides
synthesizes new DNA strand complementary
Tropoisomerase enzyme
release of tension generated by unwinding DNA
cut, unwind, together
Single-stranded DNA binding protein
prevent unwound DNA from joining reforming
protection from degradation
DNA Polymerase 1 endonuclease enzyme
- Recognises DNA/RNA hybrids removes RNA primer
2. Synthesises DNA nucleotides complementary
DNA ligase enzyme
joining of newly synthesized fragments together
forming phosphodiester bonds between okazaki fragments
Eukaryotic replication
multiple large chromosomes (vs single)
linear (vs circular)
Multiple origin of replication ori (vs single ori)
Repair DNA errors during replication
exonuclease (DNA pol 3 proofreading) 3’-5’ activity
Repair DNA errors after replication
endonuclease (removes gap) DNA polymerase fills gap
and then DNA ligase joins fragments phosphodiester
Stages of PCR
- Denaturation 94-98C
- Annealing 45-70C (primers bind to DNA template)
- Extension/Elongation 72C (DNA P adds nucleotides
- Repeat steps 2-4 25-35 times exponential increase
functions of PCR
DNA template- complementary nucleotides matched
Primers- initiate DNA synthesis 3’ OH, defines region
DNA polymerase- adds nucleotides e.g Taq
dNTPs- free nucleotides
buffer solution- maintains pH for DNA polymerase work
Divalent cations (Mg2+) cofactors essential for DNA P
