Based on the mass to charge ratio, one can identify the amino acids in a protein. Mass Spectrometry : It is used to determine the mass of particle, composition of molecule and for finding the chemical structures of molecules like peptides and other chemical compounds. After each clevage, chromatography or electrophoresis is done to identify the amino acid. A series of dark bands will appear, each corresponding to a radio labeled DNA fragment, from which the sequence can be inferred.Įdman Degradation reaction: The reaction finds the order of amino acids in a protein by cleaving each amino acid from the N-terminal without distrubing the bonds in the protein. To view the fragments, gel is exposed to X-ray film for autoradiography. Electrophoresis technique helps in arranging the fragments based on their molecular weight. This fragment is then subjected to purification before proceeding for chemical treatment which results in a series of labeled fragments. Maxam-Gilbert (Chemical degradation method): This method requires denatured DNA fragment whose 5' end is radioactively labeled. The primer or one of the nucleotides can be radioactively or fluorescently labeled also, so that the final product can be detected from the gel easily and the sequence can be inferred. Electrophoresis is done and the sequence order can be obtained by analysing the bands in the gel based on the molecular weight. When the ddNTP's gets attached to the growing chain, the chain terminates due to lack of 3'OH which forms the phospho diester bond with the next nucleotide. The 3' end of the primer accomodates the dideoxy nucleotides (specific to each tube) as well as deoxy nucleotides randomly. Next a primer is to be added which anneals to one of the strand in template. Into each of the test tubes, DNA has to be added in denatured form (single strands). Sanger Method (dideoxy chain termination method) : Here 4 test tubes are taken labelled with A, T, G and C. Similarly Edman Degradation method and Mass Spectrometry technique are used for protein sequencing. Most commonly used methods for DNA sequencing are Sanger Method and Maxam-Gilbert Method. Sequences stored in the database were obtained from different experimental methods. There are different experimental methods for sequencing, and the obtained sequence is submitted to different databases like NCBI, Genbank etc. Sequencing is the process to determine the nucleotide or amino acid sequence of a DNA fragment or a protein. Speaking biologically, a typical DNA/RNA sequence consist of nucleotides while a protein sequence consist of amino acids. Sequence is a collection of nucleotides or amino acid residues which are connected with each other. The most simpler way to answer this question is to compare their sequences. The most commonly asked question in molecular biology is whether two given sequences are related or not, in order to identify their structure or function.
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