What Distinguishes Sanger Sequencing from Edman Sequencing?
Sanger sequencing and Edman sequencing differ significantly in their principles, applications, and limitations. A detailed comparison is provided below:
Target Molecules
1. Sanger Sequencing
Designed for DNA sequencing, it identifies nucleotide sequences in nucleic acids.
2. Edman Sequencing
Tailored for protein sequencing, it focuses on analyzing the N-terminal sequence of proteins.
Principles of Sequencing
1. Sanger Sequencing
This method employs the dideoxy chain termination technique. DNA polymerase extends primers bound to a DNA template until a dideoxynucleotide (ddNTP), which lacks the 3-OH group necessary for elongation, is incorporated. This selective termination at specific bases generates DNA fragments of varying lengths, which are resolved using denaturing gel electrophoresis to deduce the sequence.
2. Edman Sequencing
This chemical degradation approach sequentially removes one amino acid residue at a time from the N-terminal of a protein or peptide. Each residue is chemically identified, enabling the reconstruction of the protein's amino acid sequence.
Detection and Sequencing Outputs
1. Sanger Sequencing
The output consists of DNA fragments of differing lengths, all sharing a common starting point but terminating at distinct nucleotides. These fragments are separated and analyzed using denaturing PAGE electrophoresis, revealing the DNA sequence.
2. Edman Sequencing
The output comprises individual amino acid residues cleaved from the N-terminal in each reaction cycle. These residues are identified using chromatographic techniques such as liquid chromatography.
Sequencing Range and Limitations
1. Sanger Sequencing
While capable of determining longer DNA sequences, its efficiency depends on factors like primer design and reaction conditions. Unknown sequences typically require prior cloning, posing challenges for large-scale genomic sequencing.
2. Edman Sequencing
Best suited for short N-terminal sequences, typically up to 40 amino acid residues. Blocked or modified N-terminal residues hinder sequencing accuracy, and the reliability diminishes with increasing sequence length.
Applications and Advantages
1. Sanger Sequencing
Commonly used for sequence validation, library screening, clone identification, and PCR product re-sequencing. Its results are straightforward and easy to interpret.
2.Edman Sequencing
A leading method for protein N-terminal sequencing, it provides direct sequence analysis without reliance on external databases, making it particularly valuable for obtaining intuitive and accurate amino acid sequences.
Sanger sequencing and Edman sequencing exhibit marked differences in their target molecules, methodologies, outputs, and limitations. The choice between these techniques should be guided by specific experimental needs and objectives.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?
