Is this a sufficient explanation of DNA sequencing:
A DNA sample is mixed with primers, DNA polymerase, nucleotides and fluorescently marked dideoxynucleotides.
PCR is performed. When a fluorescently marked nucleotide is added this terminates polymerisation, subsequently this results in many fragments differing by a single nucleotide base.
Gel electrophoresis is performed on this sample separating these fragments by the number of base pairs, the end nucleotides are tagged with a fluorescent dye and are read as peaks on a chromatogram.
And a few questions;
Why is it DNA polymerase not Taq polymerase for Dna sequencing?
In PCR are the primers/nucleotides/Taq polymerase added after heating to 95C or before? I believe it is after but textbooks explain it as a mixture before heating. My thinking is that heating to 95C would denature Taq polymerase.
Just a dot point summary:
-That is Sanger Sequencing or Chain Termination sequencing
-DNA is obtained from a sample of hair, blood etc.
- It is heated to high temperatures to separate into two single strands of DNA.
- Four flasks are set up with each base dideoxynucleotides (ddATP, ddCTP, ddGTP, ddTTP). DNA Polymerase is added not Taq DNA Polymerase because it's commercially available and you don't need Taq Polymerase as this part of Sanger sequencing doesn't occur under high heat.
- Free nucleotides are added for replication and primers for initiating replication.
- When the strand of DNA is run through each flask, each time, the DNA will keep replicating using the free nucleotides until the specific dideoxynucleotide comes up. I.e. In the flask with ddATP, it will continue to replicate until A comes up. When A does come up, the ddATP will attach to the single strand complementary and terminate the sequence.If you repeat this four times for each base, you eventually will get the exact sequence of nucleotides in the desired gene.
-When the strands are run through electrophoresis, the exact sequence of bases can be determined from the tagged ones
PCR:
Everything is added initially, but the role of primers and nucleotides are only in annealing and extension. It won't denature the enzyme. Thermus aquaticus lives in hot springs at about that temperature of 95°C (thermophile). Therefore, its enzymes have a tolerance to heat and so, won't denature at that temperature.
You seem to be on the right track Sine. I don't know how much detail is needed but yeah that's correct.