process of sanger sequencing
Sanger sequencing, also known as chain termination sequencing or dideoxy sequencing, is a method of DNA sequencing named after its developer, Frederick Sanger. It involves several steps:
1. DNA sample preparation: DNA is extracted from the organism or tissue of interest and purified.
2. PCR: Polymerase Chain Reaction (PCR) is used to amplify the DNA sample to produce many copies of the same DNA fragment. This is done by using DNA primers specific to the region of interest and DNA polymerase enzyme.
3. DNA sequencing reaction: In this step, the PCR product is used as a template in a DNA sequencing reaction. The reaction mix includes DNA polymerase enzyme, DNA primers that anneal to the template DNA, and four dideoxynucleotides (ddNTPs). These ddNTPs are labeled with different-coloured fluorescent dyes.
4. DNA sequencing analysis: After the sequencing reaction, the mixture of different-sized chain-terminated PCR products is separated by size in a polyacrylamide gel. The gel is then placed in a DNA sequencing instrument, which analyzes the fluorescent signal of each band, and identifies the nucleotide that is incorporated at that particular position in the DNA sequence.
5. Data analysis and interpretation: The instrument software generates nucleotide sequence data, which can be aligned with a reference sequence to determine the sequence of the original template DNA. The sequences are compared to reference databases to identify any mutations or variations in the sample DNA.
Overall, the Sanger sequencing process involves amplifying the target DNA, sequencing the amplified DNA, and analyzing the sequence data to determine the original DNA sequence.