In: Biology
genetics chapter 10
How does Sanger sequencing work?
Sanger sequencing or the chain termination method is a method of DNA sequencing developed by Fredrick Sanger in 1977. It is based on the principle of DNA replication. This method copies a target DNA many times to make DNA fragments of various lengths. The ends of these fragments are made by fluorescent nucleotides. The markings help to determine the sequence.
The method requires a DNA template (single-stranded), a DNA primer to act as a starter, a DNA polymerase enzyme, standard dNTPs or deoxynucleotide triphosphates (dATP, dGTP, dCTP and dTTP), and modified ddNTPs or di-deoxynucleotide triphosphates(ddATP, ddGTP, ddCTP and ddTTP). ddNTPs are different from dNTPs in that it does not have a hydroxyl group on the 3' carbon sugar ring. This means that a new nucleotide cannot be added to the existing chain and ends with ddNTPs with a dye for different bases (A, T, C, or G)
The Method:
1. Amplify the DNA sequence
2. Denature DNA using heat to produce complementary and template strands for DNA sequencing.
3. Anneal primer to 5' end of the DNA
4. Disperse the primer DNA in 4 reaction vessels
5. Add DNA polymerase and all four dNTPs
6. Add specially modified ddNTPs, one type to each vessel
7. The polymerase attaches the dNTPs to the template strand at the primer normally until a ddNTP is base-paired.
8. Once ddNTP is base-paired the sequence is terminated because of the ddNTP lacks a hydroxyl group at 3' Carbon.
9. Polyacrylamide Gel Electrophoresis is used to sequence the DNA because it has a high resolving power and can separate DNA strands that differ in length by one base pair.
10. DNA migrates from the negative pole to the positive pole in the electrophoresis plate because they have negative charges from the phosphate backbone.
11. Smaller and lighter DNA move to the bottom of the plate and results in band patterns on the plate.
12. Due to chain termination, the DNA sequences of different lengths are formed across all reaction vessels.
13. The sequence is read from the bottom to the top and results in a complementary sequence of a DNA sample.
Sanger sequencing method is highly efficient in producing high-quality DNA sequences and is used to sequence individual pieces of DNA. It is widely used for small scale sequencing on account of it being expensive and inefficient in large scale projects.