Sanger Sequencing Flashcards
Alternative name for Sanger Sequencing?
Also known as the ‘chain termination method’.
Who developed Sanger Sequencing? And when?
Frederick Sanger
1977
Aim of Sanger Sequencing?
Aim is to determine the nucleotide sequence of DNA.
What are the basic principles of Sanger Sequencing?
Synthesis of new DNA strand complementary to the target DNA.
New strand contains modified nucleotides.
Modified nucleotides halt synthesis process when incorporated.
Results in fragments of varying lengths.
What is the structure of dideoxynucleotides (ddNTPs)?
- Dideoxygenated ribose ring (sugar)
- Nucleobase
- Trisphosphate
How do ddNTPs differ in structure and function from dNTPs?
Structure: dNTP processes a 3’OH group and ddNTP does not.
Function: dNTPs build DNA sequences, while ddNTPs terminate DNA sequences
How does DNA polymerase add dNTPs?
Catalyses chemical reaction between phosphate group on new dNTP and oxygen on bound dNTP (on sugar backbone of new strand being added)
What does the binding of a new dNTP result in?
Results in the release of 2 phosphate groups.
What happens if a ddNTP is added to the strand instead of a dNTP?
Bound ddNTP has no ribose oxygen so no nucleotide can be added.
DNA chain is terminated.
What is a nucleoside?
base + deoxyribose (sugar)
Who first used Taq polymerase for Sanger Sequencing?
Vincent Murray, 1989.
What is needed for Sanger Sequencing?
- A DNA polymerase enzyme
- A primer
- The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
- ddNTPs tagged with fluorescent dye
- The template DNA to be sequenced
What is the main difference between the original vs modern Sanger Sequencing?
Original method used radioactive dyes which required X-ray film to visualise.
What are the stages of Sanger Sequencing?
- Template preparation
- Reaction Mixture
- DNA Synthesis and Termination
- Fragment Separation
- Detection and Sequence Analysis
- Data Interpretation
Why are the lengths of the DNA fragments random?
Because the ddNTPs are incorporated randomly to the new strand.
