1. DNA Sequencing LECTURE 6: Biotechnology; 3 Credit hours Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences and Technology (NUST)

2. Introduction In 1953, two researchers, namely James Watson and Francis Crick, discovered the basic structure of DNA DNA is basically a long molecule that stores coded instructions for the cell The DNA provides a basic blueprint that is responsible for the creation and functioning of cells

3. Introduction The information contained in it dictates which cells should grow and when a particular cell should die and how cells should be structured into creating various body parts For example, the DNA is responsible for determining the quality of our hair, the color and the abundance, or the lack of it

4. DNA and Nucleotides DNA stands for Deoxyribonucleic acid It is present in almost all organisms and it stores long term information that is used to construct an organic body DNA comprises of a long molecule analogous to a chain, while the links of the chain are called Nucleotides There are four different nucleotides in the DNA, namely adenine, guanine, cytosine and thymine. They are also called as “A”, “G”, “C” and “T”

5. The Need for DNA Sequencing Forensic biology uses DNA sequences to identify the organism which it is unique to To isolate the genes responsible for genetic diseases like Cystic Fibrosis, Alzheimer’s disease, myotonic dystrophy, etc., which are caused by the inability of genes to work properly Agriculture has been helped immensely by DNA sequencing It has allowed scientists to make plants more resistant to insects and pests, by understanding their genes

6. Genome Sequencing Genome sequencing is determining the exact order of DNA nucleotides in the genome In human genome 3.08 billion nucleotides are present, 99% accurately sequenced DNA sequencing includes several methods and technologies DNA sequencing can be applied in numerous fields such as diagnostic, biotechnology and biological systems The first DNA sequences were obtained in the early 1970s

7. Avery: Proposes DNA as ‘Genetic Material’ Watson & Crick: Double Helix Structure of DNA Holley: Sequences Yeast tRNA Ala 1870 1953 1940 1965 1970 1977 1980 1990 2002 Miescher: Discovers DNA Wu: Sequences Cohesive End DNA Sanger: Dideoxy Chain Termination Gilbert: Chemical Degradation Messing: M13 Cloning Hood et al.: Partial Automation Cycle Sequencing Improved Sequencing Enzymes Improved Fluorescent Detection Schemes 1986 Next Generation Sequencing Improved enzymes and chemistry Improved image processing

8. DateCost per Mb of DNA SequenceCost per Genome September-2001$5,292.39$95,263,072 March-2002$3,898.64$70,175,437 September-2002$3,413.80$61,448,422 March-2003$2,986.20$53,751,684 October-2003$2,230.98$40,157,554 January-2004$1,598.91$28,780,376 April-2004$1,135.70$20,442,576 July-2004$1,107.46$19,934,346 October-2004$1,028.85$18,519,312 January-2005$974.16$17,534,970 April-2005$897.76$16,159,699 July-2005$898.90$16,180,224 October-2005$766.73$13,801,124 January-2006$699.20$12,585,659 April-2006$651.81$11,732,535 July-2006$636.41$11,455,315 October-2006$581.92$10,474,556 Cost of DNA Sequencing

9. January-2007$522.71$9,408,739 April-2007$502.61$9,047,003 July-2007$495.96$8,927,342 October-2007$397.09$7,147,571 January-2008$102.13$3,063,820 April-2008$15.03$1,352,982 July-2008$8.36$752,080 October-2008$3.81$342,502 January-2009$2.59$232,735 April-2009$1.72$154,714 July-2009$1.20$108,065 October-2009$0.78$70,333 January-2010$0.52$46,774 April-2010$0.35$31,512 July-2010$0.35$31,125 October-2010$0.32$29,092 January-2011$0.23$20,963 April-2011$0.19$16,712 July-2011$0.12$10,497 Jan-2012 (EST)$0.09$7,950 Cost of DNA Sequencing

10. Sequencing Methods To determine the order of the nucleotide bases adenine, guanine, cytosine, and thymine in a molecule of DNA two methods were used 1.Maxam and Gilbert; Chemical Sequencing 2.Sanger; Chain Termination Sequencing These two are conventional methods Robotics and automated sequencing are based on these methods

11. 1- Maxam and Gilbert Method In 1976–1977, Allan Maxam and Walter Gilbert developed a DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases I.Chemical Modification of DNA; radioactive labeling at one 5' end of the DNA (typically by a kinase reaction using gamma- 32 P ATP) II.Purification of the DNA fragment to be sequenced III.Chemical treatment generates breaks in DNA IV.Run on the gel

12. Chemical Modification and Cleavage Ploy nucleotide Kinase radioactive label at one 5' end of the DNA using gamma- 32 P 5′ G A C G T G C A A C G A A 3′ 32 P 5′ G A C G T G C A A C G A A 3′

13. Dimethyl sulphate Purine – Adenine – Guanine Only DMS------- G DMS+ Formic acid-------G+A Piperidine Chemical Modification and Cleavage

14. DMS G G G G FA G A G G A G A A H C T T C T C C T H+S C C C C Maxam Gilbert Sequencing 32 P 5′ G A C G T G C A A C G A 3′

15. Hydrazine Pyrimidine – Cytocine – Thymidine Hydrazine----- C+T Hydrazine + NaCl--------C Chemical Modification and Cleavage

16. DMS G G G G FA G A G G A G A A H C T T C T C C T H+S C C C C Maxam Gilbert Sequencing 32 P 5′ G A C G T G C A A C G A 3′

17. Sequencing gels are read from bottom to top (5′ to 3′). G G+A T+C C 3′AGCAACGTGCAG5′3′AGCAACGTGCAG5′ Longer fragments Shortest fragments G A Maxam-Gilbert Sequencing 32 P 5′ G A C G T G C A A C G A 3′

18. 2- Sanger; Chain Termination Sequencing Its PCR based method A modified DNA replication reaction Growing chains are terminated by dideoxynucleotides

19. The 3′-OH group necessary for formation of the phosphodiester bond is missing in ddNTPs

20. ddATP + ddA fourdNTPs dAdGdCdTdGdCdCdCdG ddCTP + dAdGddC fourdNTPs dAdGdCdTdGddC dAdGdCdTdGdCddC dAdGdCdTdGdCdCddC ddGTP + dAddG fourdNTPs dAdGdCdTddG dAdGdCdTdGdCdCdCddG ddTTP + dAdGdCddT fourdNTPs dAdGdCdTdGdCdCdCdG ACGTACGT Sanger; Chain Termination Sequencing A G C T G C C C G

21. Sequencing gels are read from bottom to top (5′ to 3′) G A T C 3′GGTAAATCATG5′3′GGTAAATCATG5′ Longer fragments Shorter fragments ddG Chain Termination Sequencing