Presentation on dna sequencing

1.This presentation contain information about DNA and the mehods used for their sequencing like whole genome sequencing and shotgun sequenencing.
2.Advantages and disadvantages of whole genome sequencing and shot gun sequencing are also mentioned .
3.And the most important one is the applications of DNA sequencing.

1. I.P COLLEGE,CAMPUS II A Presentation on topic DNA Sequencing Submitted by- Meenu sharma M.Sc. biotechnology (II SEM) 1

2. WHAT IS DNA ? A.DNA stands for Deoxyribo nucleic acids. B. DNA is a molecule that carries most of the genetic instructions used in the growth, development, functioning and reproduction of all known living organisms and many viruses. C.DNA molecule consist of biopolymer strands coiled around each other to form a double helix. D.The two strands are known as polynucleotide strands since they consist of simpler units called called a nucleotides. 2

3. STRUCTURE OF A DNA NUCLEOTIDE- Each nucleotide consist of a nitrogen containing nucleo base either cytosine (c),guanine (G) ,adenine (A), or thymine (T) as well as sugar called deoxy ribose and a phosphate group .. 3

4. WHAT IS DNA SEQUENCING ? The process of determining the order of bases adenine (A), thymine (T), cytosine (C), and guanine (G) along a DNA strand . All the information required for the growth and development of an organism is encoded in the DNA of its genome. So, DNA sequencing is fundamental to genome analysis and understanding the biological processes in general . 4

5. METHODS OF DNA SEQUENCING – 5

6. A. CHEMICAL CLEAVAGE METHOD BY ALLAN MAXAM AND WALTER GILBERT (1976-1977)- Sequences DNA fragments containing upto ~500 nucleotides in length. This method uses double-stranded DNA samples. Maxam -Gilbert sequencing was the first widely used method for DNA sequencing . Involves modification of the bases in DNA followed by chemical base-specific cleavage . 6

7. STAGES - 1. The double-stranded fragment to be sequenced is isolated and radioactively labeled at the 5’-ends with 32 P. 2. The fragment is then cut with restriction enzyme and thus the label is removed from one end. 3. The fragment of DNA with one end labeled is denatured. 4. Four identical samples of these end-labeled DNA restriction fragments are subjected to chemical cleavage at different chemical nucleotides. 5. There are four specific sets of chemical reactions that selectively cut the DNA backbone at G, A+G, C+T, or C residues. G only: Dimethyl sulphate (DMS ) and piperidine A+G : DMS, piperidine C+T : Hydrazine, piperidine C only : Hydrazine, alkali, piperidine 7

8. 6 . For each labeled chain to be broken only once, the reactions are controlled. 7. The labeled sub-fragments created by the four reactions have the 32 P label at one end and the chemical cleavage point at the other end. 8. The reaction products are separated by poly acryl amide gel electrophoresis which h is based on size. Smallest fragment goes fastest 9. The labeled fragments in the gel are visualized by autoradiography. 10. The sequence is read from bottom to top of the gel. 8

9. 9

10. B.The Enzymatic method /Sanger dideoxy (Primer extension/chain termination method) The enzymatic procedure is commonly refered to as sanger-coulson method since it was developed by F.sanger and coworkers . Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain terminating dideoxy nucleotides by DNA polymerase during invitro DNA Replication. The classical chain- termination method requires – 1.A single stranded DNA template. 2.A DNA polymerase. 3.Normal dideoxynucleotide triphosphates ( ddNTP’s ). 10

11. STAGES- 1 .The DNA to be sequenced is called the template DNA . It is prepared as a single-stranded DNA after being spliced into M13 vector DNA. Infected E. coli host cells release phage particles which contains single-stranded recombinant DNA that includes the sample DNA. This DNA sample is then extracted from phage for sequencing purpose. 2 . A synthetic 5’-end-labeled oligo deoxynucleotide is used as the primer. 3 . The template DNA is hybridized to the primer. 4. The primer elongation is performed in four separate polymerization reaction mixtures. Each mixture contains - 4 normal deoxynucleotides ( dNTPs ) in higher concentration and - a low concentration of the each of the 4 ddNTPs . 5 .There is initiation of DNA synthesis by adding enzyme DNA polymeras since the enzyme cannot distinguish between the normal nucleotides and t heir analogues . 11

12. 6 . The strand synthesis continues until a ddNTP is added. The chain elongation ceases on the incorporation of a ddNTP because it lacks a 3’-OH group which prevents addition of the next nucleotide. 7 . There is a result of mixture of terminated fragments, all of different lengths. 8 . Denature DNA fragments. 9 . Each of the four mixtures are run together on a polyacrylamide gel for electrphoresis . 10 . The separated fragments are then visualized by autoradiography. 11 . From the position of the bands of the resulting autoradiogram, the sequence of the original DNA template strand can be read directly 12

13. SANGER DIDEOXY CHAIN TERMINATION METHOD 13

14. APPLICATIONS OF DNA SEQUENCING- DNA sequencing may be used to determine the sequence of individual genes , larger genetic regions ( i.e , cluster of genes or operons ),full chromosome or entire genomes . IN FORENSIC SCIENCE -DNA Sequencing has been used in forensic science-to identify particular individual because every individual has unique sequence of his/ her DNA. -to identify criminals by finding some proof from the crime scene in the form of hair,nail,skin or blood samples. -to determine paternity of the child . 14

15. IN MEDICAL RESEARCH - a .DNA sequencing can be used to detect the genes which are associated with some heredity or acquired diseases. -scientists use different techniques like gene therapy to identify the defected gene and replace them with the healthy one . IN AGRICULTURE- DNA sequencing has played a vital role in the field of agriculture. The mapping and sequencing of the whole genome of microorganisms has allowed the agriculturists to make them useful for the crops and food plants . 15

16. 16 THANK YOU FOR LISTENING ME !