Teachniques 1: seq DNA (Lucy) Flashcards
What is the role of DNA (general description)?
Makes up the majority of hertiable info across life on earth
Encodes for functional proteins, but also funtional elements (e.g. centromeres, ORI), evidence of evolutionary history, transposable elements, etc
DNA seq is a powerful method & is behind lots of otehr techniques
What is the assumed knowledge of DNA for DNA seq?
- DNA = long & biopolymer
- DNA synthesised 5’ –> 3’
- Residues are added by reaction between triphosphate of incoming dNTP & 3’-OH of new strand
- Template strand guides incorporation
Typical genome cont; bacteria = ~0.6-10Mb & Eukaryotes 10-100,000Mb
What was Sanger’s first method of DNA seq (got his first Nobel prize for)?
Chemically degrade DNA one base at a time & test which base was released (bovine insulin was used)
(Sanger was not the first to develop DNA seq, people had been succesful before him)
What was the DNA seq technique that Sanger was awarder his 2nd Nobel prize for?
Harnessing what was going on in cell division cycles - can be done in a tube
Found a method to harness power of DNA replication to read sequence –> Seq by synthesis
What is the simplified process of replication reaction in a tube (prior to Sanger seq)?
- Template (single stranded) DNA
- Add purified polymerase (E.coli DNA polymerase I)
- Synthetic primer (all the rest of the bits to replicate template strand)W
- Mix of dNTPs (deoxy nucleic acids)
What was the initial issue with DNA replication in a tube (prior to Sanger)?
Useful for replication but nood good for not good for sequencing
Can’t see which base is incorporated in which position
How did Sanger develop his method?
He looked at the basic biochem of the elongation of the chain
What was the biochemistry that Sanger studied to develop his method?
He found replication was a simple strand extension reaction
OH grp on current chain of the deoxy acid, attacks the phosphate & links the new base onto the chain
The reaction = dependent on OH grp & phosphate –> he realised if you took nucleic acids (NTPs) & removed the OH grp - converted it to a hydrogen
Why did Sanger changing the OH grp on the strand to a hydrogen for sequencing?
The hydrogen would stop the chain dead
No OH grp so it cannot attack the next base, therefore strand extension can’t occur
What did Sanger include in in his DNA replication stage of requencing to stop the chian at the correct point?
He added into the reaction a limited amt of one dideoxy NTP (ddNTP) e.g. only one of the 4 bases
(These are bases that have thier OH grp missing & replaced w H)
Incorporation stops DNA strand extension
What are the general stages of sanger sequencing?
1 - Denature DNA
2 - Anneal the primer
3 - Chain extension in presence of dideoxy nucleotide
4 - Analyse the results by polyacylamide gel electrophoresis
If we add ddATP to replication process where will the strand stop replicating?
This is an A dideoxy nucleotide so they will attach to Ts on the strand
Therefore the replication will stop at a corresponding T (chain will terminate)
What happens during the chain extension with dideoxy nucleotides stage of Sanger sequencing?
Add all of normal dNTPs & a small amt of dideoxy (in this case ddA)
OH grp is removed on these ddNTPs
How did Sanger analyse the chains he had stopped with ddNPTs?
The ddNTPs are labelled with radioactive phosphate
He put them through polyacrylamide gel electrophoresis –> 1 round for each nucleotide
