Maxam Gilbert Method
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
Maxam-Gilbert sequencing was the first widely adopted method for DNA sequencing, and, along with the Sanger dideoxy method, represents the first generation of DNA sequencing methods. Maxam-Gilbert sequencing is no longer in widespread use, having been supplanted by next-generation sequencing methods.
Procedure:
1. End Labelling- 5’ 32P Labelled ssDNA preparation
• The double stranded DNA is first denatured by heat to give single stranded DNA fragments.
• The 5’ phosphate of the ssDNA is removed and replaced by radioactive isotope 32P
• Thus all the DNA fragements in the reaction will have a radiolabelled 5’ end.
• End labelling can be done to the ds DNA, followed by denaturation and separation of ssDNA
2. Chemical treatment in 4 reaction tubes (refer to the table in video)
3. Agarose Gel electrophoresis and Autoradiography
• 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.
4. Analysis of banding pattern
• From presence and absence of certain fragments the sequence may be inferred.
Advantages
• Purified DNA can be used directly
Disadvantages
• Many steps
• Difficult to scale up (automation)