DNA sequencing Flashcards
What is the most commonly used (practical) method for DNA sequencing?
The Sanger method.
The Sanger method uses dideoxy chain terminator nucleotides. What is a dideoxy nucleotide?
The 3’ -OH group of normal nucleotides is replaced with a hydrogen.
Why does the Sanger method use dideoxy nucleotides?
The 3’ -OH group is essential for the addition of new nucleotides to a chain, as DNA is synthesised in a 5’-3’ direction, thus it prevents the addition of new nucleotides.
How many reactions are conducted simultaneously in the Sanger method, how do they differ?
4 - each reaction contains all the normal nucleotides plus a different dideoxy nucleotide, either ddATP, ddTTP, ddGTP or ddCTP.
What is needed to conduct the Sanger method?
- Target DNA: you need enough for 4 test tubes so it will first have been amplified by PCR
- DNA polymerase
- dNTPs
- ddNTPs
- Primers
Briefly explain the steps in the Sanger method.
- Run all the polymerase reactions. In each tube the DNA polymerase will construct complementary strands for the target DNA you have put in
- Put the contents of each reaction into a separate well and run an electrophoresis gel
- Visualise the gel
- Assess the banding pattern to work out the DNA sequence
How does the banding pattern produced by the Sanger method tell you the sequence of the DNA?
The chance of either a dNTP or a ddNTP being incorporated into the chain is random. If it is a dNTP the chain will stop, telling you which base sits in that position in the sequence. Fragments that travel furthest on the gel are smallest. Thus you can work out the size order of the fragments and what they end in.
Why are primers essential for the Sanger method?
It ensures that the polymerase begins synthesis from the same point on every fragment of target DNA.
To amplify the DNA for sequencing it is often cloned into a plasmid. These are self-replicated by the bacteria that possess them. True or false?
True - this is an alternative to PCR.
If the target DNA has been amplified using a plasmid, how would you design a primer for the reaction?
Find a primer that binds to the plasmid near the cloning site.
What components does automated DNA sequencing require?
The same as the Sanger method.
What does automated sequencing do that the Sanger method doesn’t? Give 3 things.
- Uses thermal cycling to obtain more copies of reaction products
- Uses fluorescently labelled ddNTPs so that only 1 reaction is necessary, not 4
- Separation uses a gel with an automated readout using a laser scanner
What kind of template DNA do you need for the Sanger method?
ssDNA.
