NUCLEOTIDE SEQUENCING
- 1. SUBMITTED TO, Dr. Monika Asthana Incharge Department Of Biotechnology SUBMITTED BY, Prashant sharma M.Sc. Biotechnology 2nd Semester
- 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.
- 17. 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.
- 18. 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.
- 19. 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
- 20. 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.