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Maxam Gilbert Sequencing

How DNA is Sequenced

DNA Sequencing using the Maxam-Gilbert Method

Maxam-Gilbert Sequencing With this method of DNA sequencing instead of synthesizing DNA in vitro and stopping the synthesis reactions with chain terminators, this method starts with full-length, end labeled DNA and cleaves it with base specific reagents. So how this method works with guanine bases, but the same principle applies to all four bases; First we end-label a DNA fragment we want to sequence.

This can be 5’- or 3’-end labeling. Next, we modify one kind of base. Here we use dimethyl sulfate (DMS) to methylate guanines. (Actually, this reagent also methylates adenines, but not in a way that leads to DNA strand cleavage.) As in the chain termination method, we do not want to affect every guanine, or we will produce only tiny fragments that will not allow us to determine the DNA’s sequence. Therefore, we do the methylation under mild conditions that lead to an average of only one methylated guanine per DNA strand. Next, we use a reagent (piperidine) that does two things: it causes loss of the methylated base, then it breaks the DNA backbone at the site of the lost base (the apurinic site). In this case, the G in the middle of the sequence was methylated, so strand breakage occurred there, producing a labeled trimer.

In another DNA molecule, the first G could not be methylated, giving rise to a labeled phosphate (the base and sugar would be lost in the chemical cleavages). Finally, we electrophorese the products and detect them by autoradiography, just as in the chain-termination method. Of course, we need to run three other reactions that cleave at the other three bases. There are several ways of doing this. For example, we can weaken the glycoside bonds to both adenine and guanine with acid; then piperidine will cause depurination and strand breakage after both As and Gs. If we electrophorese this A + G reaction beside the G only reaction, we can obtain the As by comparison. Similarly, hydrazine opens both thymine and cytosine rings, and piperidine can then remove these bases and break the DNA strand at the resulting apyrumidine sites. In the presence of 1M NaCl, hydrazine is specific for cytosine only, so we can run this reaction next to the C+T reaction and obtain the Ts by comparison.

Copyright Molecular Station 2012

 

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