Sanger Protein Sequencing Principle

Sanger protein sequencing is a widely used technique for determining protein sequences. It is based on DNA synthesis termination to identify each amino acid in a protein sequence. During DNA replication, labeled dideoxynucleotides (ddNTPs) replace normal deoxynucleotides (dNTPs). When ddNTPs are incorporated into the DNA strand by DNA polymerase, synthesis is terminated due to the lack of a 3’-OH group. Each ddNTP corresponds to an amino acid and has a unique fluorescent label. Thus, by detecting the termination positions, the order of amino acids can be determined.

 

1. Obtaining the Protein Sequence

First, a cleavage site is needed, which is a specific DNA sequence that can be cut by enzymes. Then, DNA synthesis is performed using four types of ddNTPs. Finally, capillary electrophoresis and fluorescence detection are used to determine the stopping positions of the various ddNTPs, thereby determining the order of amino acids.

 

2. Analyzing the Protein Sequence

The obtained data will show the relationship between peaks and the order of amino acids. Each peak represents an amino acid, and its position indicates its location in the protein sequence. By comparing with known amino acid sequences, the protein's sequence can be determined.

 

3. Applications

1. Protein Identification

Sanger sequencing can be used to identify unknown proteins by comparing them with known protein sequences.

 

2. Mutation Analysis

By comparing the sequences of normal and mutated proteins, mutations that lead to diseases can be identified.

 

3. Drug Design

By understanding the precise sequence of a protein, scientists can design specific drugs to interact with it.

 

4. Advantages

1. Accuracy

Sanger sequencing provides highly accurate protein sequences.

 

2. Wide Applicability

Sanger sequencing can be applied to both short and long protein fragments.

 

3. Mature and Stable

Sanger sequencing is a mature and stable technique widely used in laboratories.

 

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Protein Sequencing