Sangers method for DNA Sequencing

In these slides we are describe the method and steps of Sanger's method of DNA sequencing and the various agents which are helpful in this method.

1. Course:- Genomics Topic :- Sanger’s method for DNA sequencing

2. How are the Genome Sequenced ? The most popular method is dideoxy method also termed as sanger’s method. Method developed by :- Fred Sanger and awarded Nobel prize in 1980. Fred Sanger’s Method Step1:- To denature the double stranded DNA into the Single Stranded DNA. Step2:- In this method denatured DNA is mixed with the DNA polymerase and all four type of Bases and one of the dNTP atom is radioactive. DNA Sample All four Types of dNTPs in which one of the dNTP is radioactive atom. DNA polymerase RNA polymerase as initiator Types of dNTPs:- dATPs dCTPs dGTPs dTTPs One of them is radioactive + + +

3. Step3:- In this step the last mixture (DNA + dNTPs + DNA polymerase + RNA polymerase) is divided into the four aliquoted tubes. This mixture is divided into the 4 aliquoted tubes Tube 1 Tube 2 Tube 3 Tube 4

4. Step4:- Each tube is added a small dose of one of the four dideoxyribonucleotide triphosphate (ddNTPs = ddGTPs, ddCTPs , ddATPs , ddTTPs.). Aliquoted mixture is divided into 4 tubes and then treated with one of the radioactive ddNTPs. Tube 1 Tube 4 Tube 3 Tube 2 Tube 1 treated with radioactive ddGTPs Tube 4 treated with radioactive ddTTPs Tube 3 treated with radioactive ddATPs Tube 2 treated with radioactive ddCTPs A ddNTPs lacks the 3’ hydroxyl group (-OH) found on normal deoxyribonucleotides. When DNA is being polymerized , the newest dNTP to be added to forms a covalent bond onto the protruding 3’ hydroxyl group of the last successfully added dNTPs.

5. Step 5 :- The ddNTPs incorporated when the last dNTP is attached and there is non-availability of dNTPs in sample. The ddNTPs incorporated with the termination of elongation of DNA. Each and every DNA terminates the elongation process by incorporating with the ddGTPs Each and every DNA terminates the elongation process by incorporating with the ddTTPs Each and every DNA terminates the elongation process by incorporating with the ddATPs Each and every DNA terminates the elongation process by incorporating with the ddCTPs After incorporation of ddNTPs the reaction will stop and elongation will End.

6. Step 6:- The content of each of four types of tube is loaded into four different lenses of gel The different length of the DNA segment travels the different distance in the gel. Smallest size of DNA covers the maximum length in the gel.

7. The DNA Sequencing gel is exposed to the X- ray and read from the bottom to top respective in 5’ to 3’ direction Radioactive sequencing worked well but seems to be labor intensive it could collect only 500 bases in 24 Hours to make, run and expose to X-ray films. A faster method of sequencing was developed by Caltech, Leroy Hood and members of his lab innovate an Non-Radioactive method . They used ddNTPs and fluorescent dye with the Bases (There is no use or radioactive atom). The different ddNTPs labelled with the different color of fluorescent dye. With this innovation the time consumption in the X-ray film exposition and manual reading will be reduce. There is only tube of sample will needed to load into the Single lane of gel. Each band flashed it’s colour and indicates the presence of specific type of the ddNTPs . That data was recorded into the computer. In this method of sequencing there is replacement of the four bands into single band in the different colour flashing also termed as chromatograms.