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The genetic sequence that encodes for every protein is different, and cycle sequencing allows scientists to extract the exact DNA sequence from an unknown DNA strand. Cycle sequencing utilizes special kinds of nucleotides known as di-deoxynucleotides (along with normal deoxynucleotides) in order to get short replicas of the complimentary DNA strand of the parent DNA which has to be studied. The dideoxynucleotides (didNTP) quench the reaction because of their lack of the 3′-hydroxyl group, which is required to form the next phosphodiester linkage with the next nucleotide. The lack of this 3′-hydroxyl disables the extension of the DNA strand any further making shorter replicas of the parent molecule. The dideoxynucleotides (didNTP) are also generally labelled with a fluorescent tag. This allows the observation and characterization of the DNA sequence. The animation below demonstrates the whole process and will allow you to understand better of how cycle sequencing helps in the sequencing of the DNA.
We also recommend that you check out another animation on Sanger Sequencing – Early DNA Sequencing.
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The sequencing method developed by Fred Sanger forms the basis of automated “cycle” sequencing reactions today. Fluorescent dyes are added to the reactions, and a laser within an automated DNA sequencing machine is used to analyze the DNA fragments produced.
- To sequence a piece of DNA, you need:
- The DNA you want to sequence (template DNA)
- A short DNA “primer” that is complimentary to the DNA you want to sequence,
- An enzyme called DNA polymerase, four nucleotides.
To this mix, we also add a second type of nucleotide; one that has a slightly different chemical formula. These “dideoxynucleotides” can be recognised by a DNA sequencer.
To start the sequencing reaction, this mixture is heated, so the template DNA’s two complementary strand separate.
Then the temperature is lowered, so that the short “primer” sequence finds its complementary sequence in the template DNA.
Finally, the temperature is raised slightly. This allows the enzyme to bind the DNA and create a new strand of DNA.
The sequence of this new DNA is complimentary to the original DNA strand.
The enzyme makes no distinction between dNTPs or didNTPs. Each time a didNTP is incorporated, in this case didATP, the synthesis stops.
Because billions of DNA molecules are present in the test tube, the strand can be terminated at any position. This results in collections of DNA strands of many different lengths.
The sequencing reaction is transferred from the tube to a lane of a polyacrylamide gel.
The gel is placed into a DNA sequencer for electrophoresis and analysis.
The fragments migrate according to size , and each is detected as it passed a laser beam at the bottom of the gel.
Each type of dideoxynucleotide emits a colored light of a characteristic wavelength and is recorded band on a simulated gel image.
The computer program interprets the raw data and outputs an electropherogram with colored peaks representing each letter in the sequence.
If these are the fragments from the sequencing reaction, how would they sort out?
The simulated gel image is read from bottom to top, starting with the smallest fragment.