15.17:
Maxam-Gilbert Sequencing
In the Maxam-Gilbert, or chemical cleavage method, the template DNA is first denatured and radiolabeled at the 5' end with a Phosphorus 32.
The sequencing reaction is run in four different reaction tubes with four different chemicals with the ability to modify different nucleotides in the DNA.
For example, formic acid will only attack the purines, adenine or guanine, while hydrazine will only attack pyrimidines – cytosine or thymine. Similarly, chemicals like dimethyl sulfate will only attack a single nucleotide.
Piperidine is then used to cleave the DNA at the modified bases generating radiolabeled DNA fragments of different lengths.
The four reactions are then run parallelly on a gel and visualized by autoradiography. The sequence of the template DNA can then be deciphered by reading the autoradiograph from bottom to top
15.17:
Maxam-Gilbert Sequencing
In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their chemical-cleavage method for DNA sequencing. The Maxam-Gilbert method relies on using different chemicals that can cleave the DNA sequence at specific sites, the separation of resulting DNA fragments of variable size using electrophoresis, and deciphering the DNA sequence from the resulting gel bands.
Challenges of the Maxam-Gilbert Method
The Maxam-Gilbert Method can only be used to typically resolve up to 400bp in a single run which is comparatively lower than other traditional sequencing methods such as Sanger sequencing. Additionally, the requirement of relatively large amounts of template DNA, coupled with the use of radioisotopes and hazardous chemicals, makes the Maxam-Gilbert method less favorable for routine use. However, because of the direct use of purified DNA, the Maxam-Gilbert method is more accurate than Sanger sequencing.
Present Day Applications of Maxam-Gilbert method
Although the Maxam-Gilbert method is less widely used, it is still preferred for use in some techniques, such as DNA fingerprinting and DNA structural studies.
Suggested Reading
- Shendure J. et al. DNA sequencing at 40: past, present and future. Nature. 2017 Oct 19;550(7676):345-353. DOI: 10.1038/nature24286.