And an automatic system is used to read the fluorescent signal. Terminal Dye Sequencing reactions are run in capillary electrophoresis apparatus (no gels, hooray!). This allows conducting the reaction in one tube instead of having to do four separate reactions ( 3). Once, more different dyes are used for the different dNTPs. Now instead of using radioactive ddNTP, ddNTPs have a fluorescent dye molecule. The main components – labelled nucleotides and the gel – have just been replaced by the next technological step. However the principle of DNA extension and termination is the same. In the last 20 years CSS using radioactive nucleotides has been mostly replaced by fluorescent nucleotides. “Back when boats were made of wood and men of steel.” Eh? Terminal Dye Sanger Sequencing And with this method stretches of the same nucleotides and sequence repeats are difficult to interpret on the gel.īut despite all these troubles this is how the first DNA sequences were determined. Other problems include the single-stranded DNA looping, which causes the DNA to jump over. But this assumes your gel runs perfectly and you do not have any other problems. The sequences are read from the shortest (close to the end of the gel, as they run faster) to the longest, skipping between A, T, G and C lanes to determine your DNA sequence.Ī good CSS run will allow you to read about 300 nucleotides. Electrophoresis of the resulting DNA products on the gel results in a ladder of radioactive fragments – each fragment representing the result of a ddNTP terminating the chain at a certain nucleotide.Īfter the gel is dried, it is exposed to a X-ray film and developed. One lane for each ddNTP reaction – a lane for ddATP, ddCTP, ddGTP, and ddTTP. The infamous sequencing gel usually has four lanes. Unlike the usual dNTPs used in PCR reactions, ddNTPs don’t have 3′-OH group to form the phosphodiester bond required for chain elognation. However for Sanger sequencing you do not use just regular nucleotides, you also need modified, radioactive nucleotide called ddNTP. The classical method starts as so many PCR reactions do, with a single-stranded DNA template, a primer, DNA polymerase – and, of course, nucleotides. So know that every time you send your PCR products or plasmids for sequencing and receive a nice wavy diagram of your sequence you are looking at the results of a modified Sanger sequencing method. Now instead of reading sequencing gels we read electropherograms (Figure 1). Therefore, the first ten nucleotides were always hard to read because they run close to each other.įortunately, it’s a technique that can be (and was!) automated. The run speed of the gel should be just right for optimum separation of nucleotide sequences on the gel. Although the principle described by Sanger in 1975 sounds straightforward ( 1), sequencing gels are very long and very thin – less than a millimeter thick! They were easy to break at any stage of manipulation. I’ve never run a sequencing gel in my life, but people around me did, and they spent a lot of time on getting it just right.
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