Maxam gilbert sequencing

Описание: 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)