- DNA sequencing is the process of determining the sequence of nucleotides (As, Ts, Cs, and Gs) in a piece of DNA.
- In Sanger sequencing, the target DNA is copied many times, making fragments of different lengths. Fluorescent “chain terminator” nucleotides mark the ends of the fragments and allow the sequence to be determined.
- Next-generation sequencing techniques are new, large-scale approaches that increase the speed and reduce the cost of DNA sequencing.
What is sequencing?
Sanger sequencing: The chain termination method
Ingredients for Sanger sequencing
- A DNA polymerase enzyme
- A primer, which is a short piece of single-stranded DNA that binds to the template DNA and acts as a "starter" for the polymerase
- The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
- The template DNA to be sequenced
- Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP), each labeled with a different color of dye
Method of Sanger sequencing
Uses and limitations
- Highly parallel: many sequencing reactions take place at the same time
- Micro scale: reactions are tiny and many can be done at once on a chip
- Fast: because reactions are done in parallel, results are ready much faster
- Low-cost: sequencing a genome is cheaper than with Sanger sequencing
- Shorter length: reads typically range from nucleotides in length