Table of Contents
WHAT IS SANGER SEQUENCING?
Sanger sequencing, also known as the “chain termination system,” is a method for figuring out the order of nucleotides in DNA. It was developed in 1977 by Frederick Sanger, a two-time Nobel Laureate, and his team, giving it the name Sanger Sequence.
To understand the basic structure of DNA, refer to Figure 2.
HOW DOES SANGER SEQUENCING WORK?
Sanger sequencing can be done manually or, more commonly, using an automated sequencing machine (see Figure 1). The process involves three main steps, outlined below. Figure 1. Step-by-Step Guide to Automated Sanger Sequencing Procedures.
1. DNA SEQUENCE FOR CHAIN TERMINATION PCR
The DNA sequence of interest serves as a template for a special type of PCR known as chain-termination PCR. This process is similar to standard PCR but with a key difference – adding modified nucleotides (dNTPs) called deoxyribonucleotides (ddNTPs). In chain-termination PCR, a small amount of ddNTPs is mixed with normal dNTPs in the PCR reaction. When DNA polymerase incorporates a ddNTP, the extension stops. This results in millions to billions of oligonucleotide clones of the DNA sequence, each terminated at various lengths by 5’-ddNTPs. In homemade Sanger sequencing, four PCR reactions are set up, each with a single type of ddNTP (ddATP, ddTTP, ddGTP, and ddCTP). In automated Sanger sequencing, all ddNTPs are mixed in a single reaction, and each dNTP has a unique fluorescent marker.
2. SIZE SEPARATION IN GEL ELECTROPHORESIS
In the next step, the chain-terminated oligonucleotides are separated by size through gel electrophoresis. The DNA specimens are placed into a gel matrix, and an electrical current is employed. Since DNA is negatively charged, the oligonucleotides move toward the positive electrode based on their size. Gel electrophoresis arranges the oligonucleotides from smallest to largest. In homemade Sanger sequencing, oligonucleotides from each PCR reaction are run in separate lanes of a gel. In the process of automated Sanger sequencing, all oligonucleotides are subjected to a single capillary gel electrophoresis within the sequencing machine
3. GEL ANALYSIS & DETERMINATION OF DNA SEQUENCE
The concluding phase includes examining the gel to ascertain the DNA sequence. DNA polymerase synthesizes DNA in the 5’ to 3’ direction, and each terminal ddNTP corresponds to a specific nucleotide in the original sequence. Reading the gel bands from smallest to largest reveals the 5’ to 3’ sequence of the original DNA strand. In homemade Sanger sequencing, the operator reads all four lanes of the gel, using the lane to identify the terminal ddNTP for each band. In automated Sanger sequencing, a computer reads each band of the capillary gel, using fluorescence to identify the terminal ddNTP. This data is presented in a chromatogram, showing the fluorescent peak of each nucleotide along the template DNA.
Figure 2. DNA Structure Schematic. DNA consists of two strands forming a double helix, with each strand composed of deoxyribonucleotides (dNTPs).
HOW TO READ SANGER SEQUENCING RESULTS
Reading Sanger sequencing results depends on the DNA strand of interest and the chosen reference strand. If the reference is better for strand A, the results will be identical to strand B and vice versa. The sequence must be converted accordingly. For example, if the sequence of interest is “TACG” and the reference is suitable for that strand, the result will be “ATGC,” which needs conversion back to “TACG.” The conversion is influenced by color coding (e.g., A = pink, C = dark blue) and the dideoxynucleotide markers (T = unheroic). After electrophoresis separates the fractions, a computer reads them in order of length, producing a chromatogram with the correct sequence.
SANGER SEQUENCING VS. PCR
Sanger sequencing and PCR have similar starting materials and can complement each other. PCR amplifies entire DNA sequences, while Sanger sequencing aims to generate all possible lengths of DNA up to the full target sequence. Both methods can be combined, with PCR producing multiple clones for Sanger sequencing. This combination enhances the efficiency of the Sanger protocol, especially for longer target sequences.
