Mastering the Sanger Sequencing Protocol: A Step-by-Step Guide to Reliable DNA Sequencing

Protocol of sanger sequencing method includes

A template DNA that needs to be sequencedA short oligonucleotide primerDNA polymerase enzymeFour deoxyribonucleotides (dNTPs)

Sanger sequencing is a widely used method for DNA sequencing. The general protocol for Sanger sequencing includes the following steps:

1. Target DNA amplification: The first step in the Sanger sequencing protocol is the amplification of the target DNA fragment. This can be achieved using PCR (polymerase chain reaction) or any other DNA amplification method.

2. Purification of PCR products: Once the target DNA fragment is amplified, the PCR products need to be purified. This step is essential to remove any unwanted products, such as primers or dNTPs, that could interfere with the sequencing reaction.

3. Cycle sequencing reaction: The purified PCR products are then subjected to a sequencing reaction using fluorescently labeled dideoxynucleotides (ddNTPs), DNA polymerase, and a sequencing primer. The sequencing reaction is cycled several times, with each cycle producing fragments of different lengths terminated by one of the four ddNTPs.

4. Electrophoresis: After the cycle sequencing reaction, the fragments are separated by size using electrophoresis. The fragments are loaded onto a gel and an electric current is passed across the gel. The smaller fragments move faster through the gel, while the larger fragments move more slowly.

5. Detection and analysis: The final step in the Sanger sequencing protocol is the detection and analysis of the sequencing data. The fragments that have passed through the gel run are detected using fluorescence detectors, and the data is analyzed using dedicated software. The software identifies the sequence of the target DNA based on the pattern of fluorescent peaks produced by the ddNTPs.

In summary, the Sanger sequencing method includes target DNA amplification, purification of PCR products, cycle sequencing reaction using fluorescently labeled ddNTPs, electrophoresis, and detection and analysis of the sequencing data.

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