Question

Give the protocol and principle of maxam gillbert method of gene sequencing

Answer 1

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.[1]

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.

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 basee

Answer 2

Answer: Maxam gilber is a DNA sequencing method

https://www.youtube.com/watch?v=2WZBnuZMe2I

Refer to the video for explanation.

Explanation:

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)

Credits: Biomagica