Maxam gilbert method for DNA Sequencing

In this method we are describing the Maxam Gilbert method for the DNA Sequencing. Also, Describing the different reagents and stages of This Method and how can we read the Result of the Electrophoresis.

1. Course :- Genomics Topic:- Maxam – Gilbert method for DNA Sequencing

2. Maxam-Gilbert DNA sequencing method In this procedure the DNA sequence is break at it’s Bases which are terminally labelled DNA molecules. The labelled DNA molecule are break at the bases adenine, guanine, cytosine, and thymine with the chemical agents. Partial cleavage at each base produces a nested set of radioactive fragments extending from the labelled end of each positions of the base. Polyacrylamide gel electrophoresis resolves these single stranded DNA fragments Their size reveals in order the point of breakage. The autoradiography of a gel produced from four different chemical cleavages. Each chemical cleavage is specific for the specific base. Then these shown into bands which are easily readable. This method is only limited to resolution of polyacrylamide gel. Reagents first damage the DNA and then removed the Base from sugar. The exposed sugar is than a weak point of backbone and easily breaks. An alkali or amine catalyze series of ß – eliminations will cleave the sugar completely from it’s 3’ and 5’ side.

3. The main processes involve in Maxam-Gilbert DNA Sequencing Techniques. [ γ -32P]-ATP Synthesis Labeling 5’ Ends Labeling 3’ Ends Strand Separation Gel Elution Partial Methylation of Purines

4. Strong Guanine/Weak Adenine cleavage Strong Adenine/Weak Guanine cleavage An Alternative Guanine cleavage An alternative Strong Adenine/Weak Cytosine cleavage Cleavage at Thymine and Cytosine Cleavage Cleavage at Cytosine Reaction Time

5. Reaction Vessel Alcohol precipitation, wash and Rinse Gel Samples Gel Sequencing Autoradiography

6. For example Lac operon DNA Alu I Enzyme 64 Base pair DNA fragment It cleaves the AGCT DNA sequence Between G and C Alkaline Phosphatase Dephosphorylated DNA It removes the phosphorus from the two 5’ end of DNA [ γ -32P]-ATP It labels the both DNA end by 32P 5’ end labelled DNA with 32P

7. DNA sample Treated with alkaline phosphatase Note:- Alkaline phosphatase removes the phosphate from the terminal of DNA. ALP Treated DNA sample γ - labelled ATP with polynucleotide kinase There are two strategies to sequence the DNA 1) The double stranded DNA molecule is cut by second restriction enzyme and the two strands are resolved in polyacrylamide gel and isolated from sequencing. 2) The doubly labelled molecule is denatured and then strands are separated in gel, extracted and sequenced. Labeling of 5’ end of DNA

8. Chemistry involved in this method A) Adenine/Guanine cleavage :- Dimethyl sulfates methylates guanine at N7 position and adenine at N3. Dimethyl sulfate Guanine Methylated Guanine Adenine Dimethyl sulfate Methylated Adenine

9. The glycosidic bond of methylated purine is unstable and breaks easily on heating at neutral pH leaving sugar free. Treatment with 0.1M alkali at 90 degree then will cleave the sugar from neighboring phosphate groups. the resulting end labelled fragments are resolved in polyacrylamide gel. After resolving the fragments these can be seen into autoradiography which can be seen into the dark and light band in autoradiography. The dark bands are arise from the breakage of guanine which methylates 5 folder faster than adenine. Adenine enhanced cleavage The glycosidic bond of methylated adenosine is less stable than that of methylated of guanosine. The gentle treatment of adenosine with dilute acid release preferentially adenine. Subsequent cleavage with alkali can produce light band for adenine and dark band for guanine.

10. Cleavage at cytosine and thymine Hydrazine reacts with cytosine and thymine to cleave base and live ribo-sylurea . Hydrazine may reacts further to produce to hydrazone. After a partial hydrazinolysis in 15-18M of aqueous hydrazine at 20degree Celsius temp. the DNA cleaved with 0.5M piperidine. The final pattern contains bands of similar intensity from the cleavage at cytosine and thymine. Cleavage at cytosine:- The presence of 2M NaCl preferentially suppress the reaction with thymine and than piperidine breakages band from cytosine only.

11. Electrophoresis involve in this method The two strands are separate during electrophoresis after denaturation, on a neutral polyacrylamide gel. They can be easily excised and extracted. There are four aliquots cleavage reaction are as :- 1. Strong G 2. Weak A 3. Strong C 4. C+T There are electrophoresed at 600-1000V on a 40cm 20% Polyacrylamide/7M urea gel. After 12 hours later a second portion of each sample was loaded on gel and electrophoresis was continued.