DNA sequencing by Maxam Gilbert method |
Topic index:
1) End labeling
2) restriction enzyme digestion
3) denaturation
4) chemical degradation
5) gel electrophoresis
6) autoradiography
7) sequence determination
8) limitation
Maxam-Gilbert sequencing is a method of DNA sequencing developed by Allan Maxam and Walter Gilbert in 1976-1977. This method is based on nucleobase-specific partial chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides.
Procedure:
Maxam-Gilbert sequencing requires radioactive labeling at one 5′ end of the DNA fragment to be sequenced (typically by a kinase reaction using gamma-32P ATP) and purification of the DNA. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). For example, the purines (A+G) are depurinated using formic acid, the guanines (and to some extent the adenines) are methylated by dimethyl sulfate, and the pyrimidines (C+T) are hydrolysed using hydrazine. The addition of salt (sodium chloride) to the hydrazine reaction inhibits the reaction of thymine for the C-only reaction. The modified DNAs may then be cleaved by hot piperidine; (CH2)5NH at the position of the modified base. The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule.
The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each showing the location of identical radiolabeled DNA molecules. From presence and absence of certain fragments the sequence may be inferred.
1) Preparation of Your Sample
The DNA used in Maxam-Gilbert sequencing is first denatured into a single-stranded chain, and labeled on the 5′ end, usually with 32P.
3) Electrophoresis + Autoradiography
The next step cleaves the DNA. And this is where the Maxam-Gilbert sequencing gets really interesting. By taking advantage of piperidine and two chemicals that selectively attack purines and pyrimidines (dimethyl sulfate and hydrazine, respectively), the DNA is cleaved at specific points. To be more accurate, using different combinations of these chemicals, you can cleave a DNA sequence wherever there is a C, wherever there is a C or a T; wherever there is a G or wherever there is a G or an A. So, if you put your sample into these 4 different reaction tubes, you obtain different fragments, depending on the combination of chemicals!
3) Electrophoresis + Autoradiography
These reactions are then loaded on to a high percentage polyacrylamide gel, to differentiate fragment sizes. The fragments are visualized via the radioactive tag.
4) Reading the Sequence
To read the sequence, you begin with the smaller fragments at the bottom of the gel. “Calling” each base involves interpreting the band pattern relative to the four chemical reactions. For example, if a band in the DNA sequence appears in both the G-reaction and the G+A-reaction lanes, then that the nucleotide is a G. If a band in the DNA sequence appears only in the G+A-reaction lane, then it is an A. The same decision process works for the C-reaction and the C+T-reaction lanes. Sequences are confirmed by running replicate reactions on the same gel and comparing the autoradiographic patterns between replicates.