Lec-7 Methods of DNA sequencing.pptx amrk

DNa squencing

1. DNA Sequencing and its types A T G C……

2. What is DNA Sequencing ?

3. The process of determining the order of nucleotides adenine (A), thymine (T), cytosine (C), and guanine (G) along a DNA strand. We need to know the order of nucleotide bases in a strand of DNA for sequencing. All the information required for the growth and development of an organism is encoded in the DNA of its genome. So , DN A s equ encin g i s fundam e n t al t o g enome anal y s is and understanding the biological processes in general.

4. Classical Methods Maxam and Gilbert Sequencing method Sanger Sequencing method Advanced Methods Hierarchal shot-gun Sequencing method Whole genome Shot-gun Sequencing method Next Generation Sequencing (NGS) Pyrosequencing Sequencing by Synthesis ( Illumina ) Roche 454 Nanopore sequencing Single Molecule Real time Sequencing (SMRT)

5. Classical Methods

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

7. Advanced Methods

8. Shot-gun Sequencing method

9. Hierarchal Shot-gun Sequencing

10. 2. Whole-Genome Shot-gun Sequencing

11. Next-generation sequencing

12. NEXT GENERATION SEQUENCING (NGS) Several competing methods of Next Generation Sequencing have been developed by different companies. Pyrosequencing Sequencing by Synthesis ( Illumina ) Roche 454 Nanopore sequencing Single Molecule Real time Sequencing (SMRT)

13. 1 . Pyrosequencing Pyrosequencing is based on the ' principle where a complementary strand is synthesized in the presence of polymerase enzyme. In contrast to using dideoxynucleotides to terminate chain amplifi c a ti o n (as instead detects in San g er s e q u e ncin g ) , p y r o s e q u e n cing t h e r ele a s e o f p y r op h osph a t e whe n nucleotides are added to the DNA chain.

14. It initially uses the emulsion PCR technique to construct the colonies required for sequencing and removes the complementary strand. Next, a ssDNA sequencing primer hybridizes to the end of the strand (primer-binding region), then the four different dNTPs are then sequentially made to flow in and out of the wells over the colonies. When the correct dNTP is enzymatically incorporated into the strand, it causes release of pyrophosphate

15. In the presence of ATP sulfurylase and adenosine, the pyrophosphate is converted into ATP. This ATP molecule is used for luciferase-catalysed conversion of luciferin to oxyluciferin, which produces light that can be detected with a camera. The relative intensity of light is proportional to the amount of base added (i.e. a peak of twice the intensity indicates two identical bases have been added in succession).

16. 2. Sequencing by Synthesis ( Illumina ) 1. The first step in this sequencing technique is to break up the DNA into more manageable fragments of around 200 to 600 base pairs . 2.Short sequences of DNA called adaptors ? , are attached to the DNA fragments. 3. The DNA fragments attached to adaptors are then made single stranded. This is done by incubating the fragments with sodium hydroxide. 4. Once prepared, the DNA fragments are washed across the flowcell . The complementary DNA binds to primers ?  on the surface of the flowcell and DNA that doesn’t attach is washed away.

17. 5. The DNA attached to the flowcell is then replicated to form small clusters of DNA with the same sequence. When sequenced, each cluster of DNA molecules will emit a signal that is strong enough to be detected by a camera. 6. Unlabelled nucleotide bases and DNA polymerase ?  are then added to lengthen and join the strands of DNA attached to the flowcell . This creates ‘bridges’ of double-stranded DNA between the primers on the flowcell surface. 7. The double-stranded DNA is then broken down into single-stranded DNA using heat, leaving several million dense clusters of identical DNA sequences. 8. Primers and fluorescently- labelled terminators (terminators are a version of nucleotide base – A, C, G or T - that stop DNA synthesis) are added to the flowcell .

18. 9. The primer attaches to the DNA being sequenced. 10. The DNA polymerase then binds to the primer and adds the first fluorescently- labelled terminator to the new DNA strand. Once a base has been added no more bases can be added to the strand of DNA until the terminator base is cut from the DNA. 11. Lasers are passed over the flowcell to activate the fluorescent label on the nucleotide base. This fluorescence is detected by a camera and recorded on a computer. Each of the terminator bases (A, C, G and T) give off a different colour . 12. The fluorescently- labelled terminator group is then removed from the first base and the next fluorescently- labelled terminator base can be added alongside. And so the process continues until millions of clusters have been sequenced.

19. 13. The DNA sequence is analysed base-by-base during Illumina sequencing, making it a highly accurate method. The sequence generated can then be aligned to a reference sequence, this looks for matches or changes in the sequenced DNA. 

20. Steps involved in Illumina

21. 3. Roche 454

22. 4. Nanopore sequencing

23. INTRODUCTION The fourth-generation DNA sequencing technology Studies the interaction between DNA and protein, as well as between protein and protein. Have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. The detection principle is based on monitoring the ionic current passing through the nanopore as a voltage is applied across the membrane. When the nanopore is of molecular dimensions, passage of molecules (e.g., DNA) cause interruptions of the current level, leading to a signal .

24. In the 1990s, Church et al. And Deamer and Akeson separately proposed that it is possible to sequence DNA using nanopore sensors

25. Nanopore technologies can be broadly divided into two categories: Biological nanopore Solid-state nanopore More recently, hybrid nanopores have been proposed to take advantage of the features of both biological and solid-state nanopores. TYPES OF NANOPORES

26. 5. Single Molecule Real time Sequencing (SMRT) Zero-mode waveguides allow real-time observation f single molecule at high concentration. They are photonic nanostructures that optically observed single fluorescent molecule.