Sanger Sequencing
Sanger sequencing, aka dideoxy chain-termination sequencing, is a method for determining the nucleotide sequence of DNA. Developed by Frederick Sanger in 1977, it revolutionized molecular biology and genomics by providing a reliable and relatively simple way to read DNA sequences.
One of the most important components of Sanger sequencing is the use of ddNTPS (dideoxynucleotide triphosphates). These nucleotides, unlike regular dNTPs, lack a 3' hydroxyl group, which causes DNA chain termination when incorporated during DNA synthesis. This property is utilized in Sanger sequencing.
Process:
Denaturing of the copies of the fragment of interest into single strands
Incubation of the strands with ingredients for DNA synthesis: specific primer, DNA polymerase, dNTPs, and ddNTPS
As DNA polymerase synthesizes the new DNA strands, it incorporates both dNTPS and ddNTPs
The ddNTPs lack a 3' hydroxyl group, which prevents further elongation of the DNA strand once incorporated
Each ddNTP is labeled with a distinct fluorescent dye or radioactive tag, allowing identification of the terminating nucleotide
The resulting fragments of varying lengths are separated by size using gel or capillary electrophoresis.
The sequence is determined by analyzing the pattern of termination.
The process produces a mixture of DNA fragments of varying lengths, each terminating at a different position corresponding to the incorporation of a ddNTP. These fragments are then separated by size using gel or capillary electrophoresis, and the terminating nucleotides are detected through their fluorescent labels. By analyzing the sequence of fluorescent signals, the original DNA sequence can be determined.
Problem:
The following gel has resulted after Sanger sequencing. Using your understanding of the procedure, identify the original sequence of the DNA fragment.
Solution:
A Sanger sequencing gel consists of lanes corresponding to the four different nucleotides (A, T, C, G). The bands represent DNA fragments terminated at different positions by the incorporation of dideoxynucleotides. To determine the DNA sequence, you read the gel from bottom to top, starting with the smallest fragments (which ran the furthest) to the largest fragments (which ran the least).
Interpreting the Gel:
Identify the Lanes: Each lane represents a different nucleotide (A, T, C, G).
Read from Bottom to Top: Begin at the bottom of the gel where the smallest fragments are located and read upwards to determine the sequence of nucleotides.
Sequence Determination: Write down the nucleotide corresponding to each band, moving from the bottom to the top of the gel.
Following this approach, the sequence read from bottom to top is:
A, T, G, C, C, A, G, T, A
Author: Sanjay Adireddi
References: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727787/
