Difference between sanger sequencing and dna repkication
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Answer:
What is Sanger Sequencing?
Sanger sequencing, also known as the “chain termination method”, is a method for determining the nucleotide sequence of DNA. The method was developed by two time Nobel Laureate Frederick Sanger and his colleagues in 1977, hence the name the Sanger Sequence.
Sanger Sequencing Steps
There are three main steps to Sanger sequencing.
What is DNA replication?
DNA replication is the process by which DNA makes a copy of itself during cell division.
1. The first step is generating n DNA fragments of varying lengths, each terminated with a labeled nucleotide, where n is the number of nucleotide bases in the target DNA sequence. This is done by combining DNA primer, nucleotides (dATP, dCTP, dGTP, dTTP), DNA polymerase, the DNA sequence of interest, and labeled dideoxynucleotides (ddATP, ddCTP, ddGTP, ddTTP). No nucleotide can be added to the DNA chain once a dideoxynucleotides has been incorporated, so each fragment will end with a labeled nucleotide. A much smaller amount of dideoxynucleotides is used than the amount of regular nucleotides.
2. The second step is to separate the n DNA sequences be length using capillary gel electrophoresis. The shorter fragments move faster than the longer fragments. The result is that the DNA pieces are fed into the third step from shortest to longest sequence.
3. In the third step a laser excites the label on the nucleotide at the end of each sequence. Each base is tagged with a different label, so the light emitted by each excited nucleotide can be tied to the correct base. The laser generates a chromatogram showing the fluorescent peak of each nucleotide. The chromatogram has the nucleotides in the correct order because of the electrophoresis.
The separation of the two single strands of DNA creates a ‘Y’ shape called a replication ‘fork’. The two separated strands will act as templates for making the new strands of DNA.
One of the strands is oriented in the 3’ to 5’ direction (towards the replication fork), this is the leading strand?. The other strand is oriented in the 5’ to 3’ direction (away from the replication fork), this is the lagging strand?. As a result of their different orientations, the two strands are replicated differently:
Illustration showing replication of the leading and lagging strands of DNA.
An illustration to show replication of the leading and lagging strands of DNA.
Image credit: Genome Research Limited
Leading Strand:
A short piece of RNA ?called a primer? (produced by an enzyme called primase) comes along and binds to the end of the leading strand. The primer acts as the starting point for DNA synthesis.
DNA polymerase? binds to the leading strand and then ‘walks’ along it, adding new complementary? nucleotide? bases (A, C, G and T) to the strand of DNA in the 5’ to 3’ direction.
This sort of replication is called continuous.
Lagging strand:
Numerous RNA primers are made by the primase enzyme and bind at various points along the lagging strand.
Chunks of DNA, called Okazaki fragments, are then added to the lagging strand also in the 5’ to 3’ direction.
This type of replication is called discontinuous as the Okazaki fragments will need to be joined up later.
Once all of the bases are matched up (A with T, C with G), an enzyme called exonuclease strips away the primer(s). The gaps where the primer(s) were are then filled by yet more complementary nucleotides.
The new strand is proofread to make sure there are no mistakes in the new DNA sequence.
The result of DNA replication is two DNA molecules consisting of one new and one old chain of nucleotides. This is why DNA replication is described as semi-conservative, half of the chain is part of the original DNA molecule, half is brand new.