Question

Chemical for adenine cleave out in maxam gilbert method

Answer 1

This chapter discusses the Maxam–Gilbert chemical procedure for DNA sequencing. Since its introduction in 1977, this procedure has become the most widely used sequencing procedure for DNA. In the chemical DNA sequencing method, end-labeled DNA is partially cleaved at each of the four nucleotide bases in four different reactions, the products ordered by size by gel electrophoresis and the sequence read-off an autoradiograph by noting the base-specific agent that cleaved at each successive nucleotide along the strand. No in vitro enzymatic copying is required and either single- or double-stranded DNA, labeled at either the 3′- or 5′-end, can be sequenced. In case of Maxam–Gilbert procedure, the crucial step is the specific chemically-induced cleavage at one or two of the four nucleotide bases. This is a three-stage process, involving modification of the base, removal of the modified base from its sugar, and finally strand scission at that sugar. Dimethylsulphate is an alkylating agent that methylates ring nitrogens in the DNA bases. As far as sequencing is concerned, the relevant methylation sites are the N7 position in guanine and the N3 in adenine.

Answer 2

Answer: For cleaving Adenine+Guanine-Dimethylsulfate+ high temperate + NaOH is used

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