Basic DNA Sequencing Flashcards
What did Maxam and Gilbert’s ‘wandering spot’ method involve?
It involved laborious chromatography to define nucleotide sequence.
What did Maxam and Gilbert’s ‘chemical method’ of DNA sequencing involve?
Their chemical method involved breaking up labelled DNA chemically so that you could run the fragments on gel electrophoresis and work out from the size which base was at which position in the sequence. It was a very hazardous method.
Chemical, sequencing was based on nucleobase specific partial chemical modification of the DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides.
Cleaving of the same tagged segment of DNA at different points yields fragments of different sizes. The fragments may then be separated by gel electrophoresis.
Describe the Sanger chain termination method of DNA sequencing.
The chain termination method involved synthesis of new DNA using a template sequence that you wanted to know the base sequence of, but incorporating 2’3’ dideoxy-nucleotide triphosphates (ddNTPs) as chain terminators.
Fragments could then be separated by size (in separate lanes for each nucleotide) and you could build up the sequence by examining the terminating base in each fragment.
ddNTPs terminates the DNA synthesis at each point where that particular nucleotide occurs on the DNA sequence so you can tell where the nucleotides occur from this.
The dideoxy NTPs lack the 3’ OH groups that are required to build the nucleotide chain. The molecule can be incorporated into DNA but cannot provide the OH group for chain extension thereafter so it halts DNA synthesis and terminates the chain.
Chemical and Sanger methods require use of 32P (or 33P or 35S) labelling of either primers of of a dNTP, 4 lanes of electrophoresis per sample.
How has the chain termination method since been modified?
The chain termination method has been improved by the advent of fluorescent ddNTPs and automated sequencing methods.
The use of fluorescent dye tags on the ddNTPs was critical in the development of automated methods.
The fluorescence allowed for a ‘one tube’ methodology rather than having to run each radio labelled nucleotide in separate tubes and lanes.
All NTPs and all equivalent ddNTPs can be mixed in the same tube. ddNTPs are at a very low concentration so that there is only a tiny chance of a chain terminator being incorporated at each point in the chain. Eventually you will get a ddNTP incorporated at each point that there is the equivalent dNTP usually incorporated in the sequence. Each ddNTP has a different coloured dye attached.
All possible terminations are achieved which are then separated by electrophoresis and the fluorescence is read thus allowing the sequence to be read.
The accuracy of machine reading of DNA is very good.
Describe automated sequencing.
Automated sequencing machines can carry out many simultaneous sequencing reactions (16x-96x).
There is automation of preparation, loading sample, electrophoresis, reading sequence and matching to known sequence.
A single HTP machine can sample approximately 1 mill bases per day.
NGS methods can generate more than 20 billion bases per run.
The current aim is to be able to sequence an entire genome for $1000 as this will be the start of truly personalised medicine.
In what year was Sanger sequencing developed?
1978.
