NUCLEOTIDE SEQUENCING

ppt on nucleotide sequencing

1. SUBMITTED TO, Dr. Monika Asthana Incharge Department Of Biotechnology SUBMITTED BY, Prashant sharma M.Sc. Biotechnology 2 nd Semester NUCLEOTIDE SEQUENCING

2. WHAT IS DNA DNA is the molecule that is the hereditary material in all living cells. Genes are made of DNA. A gene consists of enough DNA to code for one protein, and a genome is simply the sum total of an organism's DNA. The DNA has four bases i.e ; Adenine (A) Thymine (T) Cytosine (C) Guanine (G)

3. WHAT IS DNA SEQUENCING DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases A,T,G &C in a strand of DNA

4. HISTORY The sequencing of DNA molecules began in the 1970s with development of the MaxamGilbert ethod , and later the Sanger method. Originally developed by Frederick Sanger in 1975. 1987 – Applied biosystems marketed Fully automated sequencing machines

5. METHOD’S OF DNA SEQUENCING Historically , there are two main methods i.e ; 1- Maxam Gilbert method or Chemical cleavage method 2- Sangers method or chain termination method Some other sequencing methods are – 1- Automated sequencing 2- Whole genome sequencing 3- Next generation sequencing

6. MAXAM GILBERT SEQUENCING METHOD By A.M. Maxam and W.Gilbert-1977 Chemical sequencing Treatment of DNA with certain chemicals DNA cuts into fragmentsmonitoring of sequences

7. PRINCIPLE The partially cleaved DNA fragment is subjected to five separate chemical reactions. Each of which is specific for a particular base. The resulting fragment terminate at that specific base followed by high resolution gel electrophoresis and detection of the labeled fragments by autoradiography

8. METHOD Labeled DNA at one end with 32P. DNA copies are divided in to 4 samples. Each samples treated with a chemical that specifically destroys one or two of the 4 base in DNA.

9. PROCEDURE

10. SANGER’S METHOD Most common approch used for DNA sequencing. Invented by “Frederick Sanger” in 1977. Noble prize in -1980. Also termed as Chain termination method or Dideoxy method.

11. PRINCIPLE Enzymatic synthesis of complementary polynucleotide chains Termination at specific nucleotide positions Separate by Gel/Capillary Electrophoresis Read DNA sequence

12. REQUIREMENTS Single Stranded template Primer DNA polymerase Di- Deoxynucleotide The 3′-OH group necessary for formation of the phosphodiester bond is missing in ddNTPs ) Every nucleotide have its specific ddNTP form i.e., ddATP , ddGTP etc

13. METHOD Steps: 1. Denaturation 2. Primer attachment and extension of bases 3. Termination 4. Gel electrophoresis

14. PROCEDURE

15. AUTOMATED SEQUENCING METHOD Florescence technique is used for detection of DNA bands. Fluorescent labbeled dd NTP are used . Resulting DNA strands are seperated in 4 different lane in electrophoresis. Detected by fluorescence detector.

16. WHOLE GENOME SEQUENCING METHOD SHORTGUN SEQUENCING METHOD Shotgun sequencing, also known as shotgun cloning, is a method used for sequencing long DNA strands or the whole genome.

17. NEXT GENERATION SEQUENCING METHODS The next generation sequencing is based upon the principle of Sanger’s method . There are many types of NGS are present I.e ; 1- Pyrosequencing 2- Illumina sequencing 3- Solid sequencing etc.

18. ADVANTAGES / DISADVANTAGES   ADVANTAGES   1) Improved diagnosis of disease . 2) Bio pesticides . 3) Identifying crime suspects . 4) Sequencing the whole Genome of organisms (e.g. Human genome project) .   DISADVANTAGES   1) Whole genome cannot be sequenced at once . 2) Very slow and time consuming

19. APPLICATIONS Forensics ;- to help identify individuals because each individual has a different genetic sequence. . Medicine;- can be used to help detect the genes which are linked to various genetic disorders such as muscular dystrophy. Agriculture;- The mapping and sequencing of a genome of microorganisms has helped to make them useful for crops and food plants.

20. THANK YOU THANK YOU