Dna sequencing methods sudha's
- 1. DNA SEQUENCING By : Kalluru Sudha Mani ADPM/17/2555 Sudha's KSM 95
- 2. Dr. Balasaheb Sawant Konkan Krishi Vidhyapeth COLLEGE OF AGRICULTURE 2017-18 PRINCIPLES OF BIOTECHNOLOGY COURSE INSTRUCTOR: Dr .S.V. SAWARDEKAR. Sudha's KSM 95
- 3. • Determining the precise order of nucleotides in a segment of DNA. Sudha's KSM 95
- 4. DNA sequencing methods: • Rapid and efficient – EARLIER METHODS – 1.Maxam & Gilbert at Harvard (1977) 2.Sanger & Coulson at Cambridge (1977) High through put DNA sequencing. Sudha's KSM 95
- 5. • Chemical degradation of the original DNA. • Treatment of DNA with certain Chemicals • DNA cuts into Fragments • Monitoring of sequences Sudha's KSM 95
- 8. • Most common approach used for gene sequencing. • Nobel prize – 1980, 1958 (INSULIN SEQUENCE). • Also known as chain termination or Dideoxy method. • Materials required: PRIMER (RADIO ACTIVE) TEMPLATE DNA dNTPs (deoxy NTP) ddNTPs (dideoxy NTP) Sudha's KSM 95
- 10. What happens when ddNTP is incorporated?? Sudha's KSM 95
- 11. Steps: 1. Many copies of DNA fragments are made. (PCR) 2. Fragments are heated again to be unwounded in to single stranded DNA. 3. Primer is added which binds to the DNA. 4. ddNTPs are added. Sudha's KSM 95
- 12. ss DNA with primer: Sudha's KSM 95
- 13. Addition of ddATP: Sudha's KSM 95
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- 16. • The newly synthesized strands of DNA have different lengths depending on when a ddNTP was incorporated into the strand. • The process is repeated 4 times with different ddNTPs. Sudha's KSM 95
- 17. Each reaction vessel only has one of the 4 ddNTP’s. Sudha's KSM 95
- 18. The strands of different lengths will be separated using gel electrophoresis onto 4 lanes one for each ddNTP. Sudha's KSM 95
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- 21. REAL SEQUENCING GEL Sudha's KSM 95
- 22. • SAME AS SANGER SEQUENCING. • 4 different coloured fluorescent dyes used to label the 4 different ddNTPs in a single testtube. • All the fluorescent ddNTPs can be run on one lane on a gel w Sudha's KSM 95
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- 25. COMPLETE GENOME SEQUENCING PROJECTS: • 1995- In Haemophius influenzae. • 1997 – in E.coli. • 2001 – human genome was sequenced successfully. - Laying foundation of genomics. • 2005 – Rice genome sequenced under International Rice Genome Sequencing Project. Sudha's KSM 95
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