BGM1002/L15 DNA Sequencing Flashcards
Describe initiation of pre-priming.
DnaA assembly stimulates unwinding of AT-rich array and recruits helicase (DnaB)
Describe loading of pre-priming. (2)
DnaB closure around ssDNA
DnaC hydrolyses ATP and dissociates
What is the function of DnaG primase?
Synthesis of RNA primer
What is DnaG primase recruited by?
DnaB
Give 3 functions of Topoisomerase I.
Catalyses relaxation of supercoiled DNA
Cleavage of one strand
Passage of cut end under other strand
Resealing the break
Give 3 functions of Topoisomerase II.
Catalyses untangling of DNA duplexes
Cleavage of both strands
Passage separate duplex molecule through break
Resealing the break
Give the 3 domains of the polymerase alpha unit.
Fingers
Palm
Thumb
What is the function of the polymerase exonuclease domain?
3’-5’ proofreading
What is the function of the sliding clamp? (3)
Accommodates dsDNA
Keeps polymerase III in contact with DNA
Increases processivity of polymerase III
Describe the lagging strand problem.
DNA is synthesised 5’-3’
Nonsense strand runs 3’-5’ so is synthesised in Okazaki fragments
Explain how coordinated synthesis of leading and lagging strands occurs.
Two DNA polymerases follow a single DnaB helicase
Describe the Trombone model.
Looping of lagging strand
Lets go after 1000nts
New loop formed
Loop lengthens and shortens like a trombone
What does the term ‘homologous recombination’ mean?
If one strand of DNA is damaged, the other strand can be used to repair it.
What experiment did Sanger perform in 1977?
Dideoxy sequencing
Describe the Sanger experiment (1977).
Gel electrophoresis with fluorescent ddNTPs
Sequence read from gel electrophoresis
