Disadvantages of this method include :

⇒  Once we get the N terminal amino acid, the protein is already hydrolyzed in constituent amino acids. Thus we cannot repeat the cycle with same sample. For second amino acid sequencing we require new stock of protein sample and the N-terminal residue need to be cleaved from the protein using an appropriate protease such as amino peptidase. This makes the process very tedious and complicated.

⇒  These dyes selectively labels the amine groups present in the protein and therefore can label the amine groups present in the side chains as well, which may give erroneous results.

Protein Sequencing using Molecular Biology techniques

If first few N-terminal amino acid of a protein is known, complete aminoacid sequence can be derived using Molecular Biology techniques. A simple example is as follow:

The genome sequence of Calotropis procera, a plant, or the sequence of procerain B, a novel cystein protease from the plant, gene is not yet known. Thus, the only information for cloning of cDNA we have is the fifteen N-terminal amino acid residues. The double stranded cDNA can be amplified with help of degenerate primer (based of N-terminal amino acid sequence) and oligo dT primer. Total RNA can be isolated from young leaf or latex of the plant and first strand of cDNA can be synthesised with oligo dT primer by reverse transcription. The second strand of cDNA can be synthesised and the subsiquent amplification of double stranded cDNA can be achived by PCR with degenerate primer as forward and oligo dT primer as reverse primmer. The amplified double stranded cDNA of expected size can be subjected to TA cloning and confirmed by sequencing. Once sequence of cDNA is availabe, it can be translated in protein sequnce.

[We shall study Few Molecular Biology techniques during coming lectures]