Dna sequencing

Sequencing Methods

1. DNA Sequencing Dr Ravi Kant Agrawal , MVSc , PhD Senior Scientist (Veterinary Microbiology) Food Microbiology Laboratory Division of Livestock Products Technology ICAR-Indian Veterinary Research Institute Izatnagar 243122 (UP) India

2. Nucleic acid sequencing The term DNA sequencing refers to  sequencing  methods for determining the order of the  nucleotide  bases - adenine ,  guanine ,  cytosine , and  thymine in a molecule of  DNA . All the information required for the growth and development of an organism is encoded in the DNA of its genome. So, DNA sequencing is fundamental to genome analysis and understanding the biological processes in general. The advent of DNA sequencing has significantly accelerated biological research and discovery. Knowledge of DNA sequences has become indispensable for basic biological research, and in numerous applied fields such as diagnostics,  biotechnology ,  forensic biology  and biological  systematics . The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of the  human genome , in the  Human Genome Project .

3. Technical Breakthrough For DNA Sequencing In 1977, two separate methods for the large-scale sequencing of DNA were devised : Chemical cleavage method by A. M. Maxam and W. Gilbert Enzymatic chain termination method by F. Sanger et. al. Of these two methods, Sanger method is more popular. Without changing the underlying concept of both methods, some improvements have been done over the years by applying different strategies, by developing various modifications and by automation. As a result, a very large scale sequencing has become feasible, e.g. E. coli , Saccharomyces cerevisiae , Human Genome Project etc.

4. Maxam –Gilbert Sequencing In 1976–77, Allan Maxam  and  Walter Gilbert   developed a DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases. This method is sometimes called Chemical Sequencing.  Maxam –Gilbert sequencing rapidly became more popular, since purified DNA could be used directly, while the initial Sanger method required that each read start be cloned for production of single-stranded DNA. However, with the improvement of the chain-termination method, Maxam -Gilbert sequencing has fallen out of favour due to its technical complexity prohibiting its use in standard molecular biology kits, extensive use of hazardous chemicals, and difficulties with scale-up.

5. Cont… The method requires radioactive labelling at one end and purification of the DNA fragment to be sequenced. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Thus a series of labelled fragments is generated, from the radiolabelled end to the first "cut" site in each molecule. The fragments in the four reactions are arranged side by side in  gel electrophoresis  for size separation. To visualize the fragments, the gel is exposed to  X-ray film  for  autoradiography , yielding a series of dark bands each corresponding to a  radiolabelled  DNA fragment, from which the sequence may be inferred.

6. Chemical Cleavage Method This method uses double-stranded DNA samples. Involves modification of the bases in DNA followed by chemical base-specific cleavage. Sequences DNA fragments containing upto ~500 nucleotides in length.

7. The double-stranded fragment to be sequenced is isolated and radioactively labeled at the 5’-ends with 32 P. The fragment is then cut with restriction enzyme and thus the label is removed from one end. The fragment of DNA with one end labeled is denatured. Four identical samples of these end-labeled DNA restriction fragments are subjected to chemical cleavage at different chemical nucleotides . There are four specific sets of chemical reactions that selectively cut the DNA backbone at G, A+G, C+T , or C residues. G only: Dimethyl sulphate ( DMS) and piperidine A+G : DMS, piperidine C+T : Hydrazine, piperidine C only : Hydrazine, alkali, piperidine Figure: Maxam -Gilbert method ( continued) Lodish , H.;Berk , A. et. al. (4 th ed ); Mol . Cell Biol .; W. H. Freeman and Co. ( 2000) p : 233 Stages

8. Maxam -Gilbert sequencing is performed by chain breakage at specific nucleotides. 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

9. 6.For each labeled chain to be broken only once, the reactions are controlled. 7.The labeled sub-fragments created by the four reactions have the 32 P label at one end and the chemical cleavage point at the other end. 8.The reaction products are separated by polyacrylamide gel electrophoresis which is based on size. Smallest fragment goes fastest. Figure: Apparatus for gel electrophoresis Voet , D.; Voet , J. and Pratt, C. (upgrade ed ) Fundamentals of Biochemistry ; John Wiley and Sons, Inc (2002); p: 58 Contd …

10. 9. The labeled fragments in the gel are visualized by autoradiography. 10. The sequence is read from bottom to top of the gel. Figure: Maxam -Gilbert method Lodish , H.;Berk , A. et. al. (4th ed ); Mol . Cell Biol .; W. H. Freeman and Co. ( 2000) p : 233

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

12. Example of DNA Sequencing by Chemical Method http://users.wmin.ac.uk/~redwayk/lectures/sequence.htm

13. Mechanism of the chemical cleavage method Voet , D.; Voet , J. Biochemistry ; John Wiley and Sons, Inc (1990); p: 830

14. Continued…. Voet , D.; Voet , J. Biochemistry ; John Wiley and Sons, Inc (1990); p: 831

15. Advantages Disadvantages No premature termination due to DNA sequencing. So, no problem with polymerase to synthesize DNA. Stretches of DNA can be sequenced which can not be done with enzymatic method. Not widely used. Use of radioactivity and toxic chemicals. http://www.cmb.uab.edu/courses/lectures/scheirer2.pdf

16. Chain-termination methods The chain-terminator method (or Sanger method after its developer  Frederick Sanger ) is more efficient and uses fewer toxic chemicals and lower amounts of radioactivity than the method of Maxam and Gilbert, it rapidly became the method of choice. The key principle of the Sanger method was the use of  dideoxynucleotide   triphosphates ( ddNTPs ) as DNA chain terminators.

17. The classical chain-termination method requires a single-stranded DNA template, a DNA  primer , a  DNA polymerase , radioactively or  fluorescently  labeled nucleotides, and modified nucleotides that terminate DNA strand elongation. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard  deoxynucleotides  ( dATP , dGTP , dCTP and dTTP ) and the  DNA polymerase . To each reaction is added only one of the four dideoxynucleotides  ( ddATP , ddGTP , ddCTP , or ddTTP ) which are the chain-terminating nucleotides, lacking a 3'- OH  group required for the formation of a phosphodiester bond  between two nucleotides, thus terminating DNA strand extension and resulting in DNA fragments of varying length.

18. The newly synthesized and labeled DNA fragments are heat  denatured , and separated by size (with a resolution of just one nucleotide) by  gel electrophoresis  on a denaturing  polyacrylamide -urea gel with each of the four reactions run in one of four individual lanes (lanes A, T, G, C). The DNA bands are then visualized by  autoradiography  or UV light, and the DNA sequence can be directly read off the  X-ray film  or gel image. In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide ( ddATP , ddGTP , ddCTP , or ddTTP ).

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

20. Chain Termination method This method uses single-stranded DNA. Also known as dideoxy sequencing method because it involves the use of analogue of normal nucleotide 2’,3’-dideoxynucleoside triphosphates ( ddNTPs ). These are chain terminating nucleotides lacking 3’-OH ends . This method is based upon the incorporation of ddNTPs into a growing DNA strand to stop chain elongation. Figure: Structure of NTP, dNTP , and ddNTP Lodish , H.;Berk , A. et. al. (4th ed ); Mol . Cell Biol .; W. H. Freeman and Co. (2000), p: 233

21. Chain terminates at ddG Chain Termination (Sanger) Sequencing The 3 ′ -OH group necessary for formation of the phosphodiester bond is missing in ddNTPs .

22. Chain Termination (Sanger) Sequencing With addition of enzyme (DNA polymerase), the primer is extended until a ddNTP is encountered. The chain will end with the incorporation of the ddNTP . With the proper dNTP:ddNTP ratio, the chain will terminate throughout the length of the template. All terminated chains will end in the ddNTP added to that reaction . The collection of fragments is a sequencing ladder. The resulting terminated chains are resolved by electrophoresis. Fragments from each of the four tubes are placed in four separate gel lanes.

23. Stages: 1.The DNA to be sequenced is called the template DNA. It is prepared as a single-stranded DNA after being spliced into M13 vector DNA. Infected E. coli host cells release phage particles which contains single-stranded recombinant DNA that includes the sample DNA. This DNA sample is then extracted from phage for sequencing purpose. 2.A synthetic 5’-end-labeled oligodeoxynucleotide is used as the primer. 3.The template DNA is hybridized to the primer. 4. The primer elongation is performed in four separate polymerization reaction mixtures. Each mixture contains 4 normal deoxynucleotides ( dNTPs ) in higher concentration and a low concentration of the each of the 4 ddNTPs . 5.There is initiation of DNA synthesis by adding enzyme DNA polymerase since the enzyme cannot distinguish between the normal nucleotides and t heir analogues. Figure: Action of DNA polymerase I Voet , D.; Voet , J. and Pratt, C. (upgrade ed ) Fundamentals of Biochemistry ; John Wiley and Sons, Inc (2002); p: 60

24. 6.The strand synthesis continues until a ddNTP is added. The chain elongation ceases on the incorporation of a ddNTP because it lacks a 3’-OH group which prevents addition of the next nucleotide. 7.There is a result of mixture of terminated fragments, all of different lengths. 8. Denature DNA fragments. 9. Each of the four mixtures are run together on a polyacrylamide gel for electrphoresis . Figure: Sanger method Lodish , H.;Berk , A. et. al. (4th ed ); Mol . Cell Biol .; W. H. Freeman and Co. ( 2000) p : 234

25. 10.The separated fragments are then visualized by autoradiography . 11. From the position of the bands of the resulting to radiogram , the sequence of the original DNA template strand can be read directly. Figure: Chain termination method Voet , D.; Voet , J. and Pratt, C. (upgrade ed ) Fundamentals of Biochemistry ; John Wiley and Sons, Inc (2002); p : 61

26. Advantages Disadvantages Most popular method. Simpler and quicker allowing large output. Within an hour the primer-annealing and sequencing reactions can be completed. Yielding of poor results owing to secondary structure in the DNA as sometimes DNA polymerases terminate chain elongation prematurely. The sequence is obtained not from the original DNA molecule but from an enzymatic copy. So , there is a chance of incorporation of wrong bases.

27. Example of DNA Sequencing in Sanger Method http://users.wmin.ac.uk/~redwayk/lectures/sequence.htm

28. Cycle Sequencing Cycle sequencing is chain termination sequencing performed in a thermal cycler. Cycle sequencing requires a heat-stable DNA polymerase.

29. Fluorescent Dyes Fluorescent dyes are multicyclic molecules that absorb and emit fluorescent light at specific wavelengths. Examples are fluorescein and rhodamine derivatives. For sequencing applications, these molecules can be covalently attached to nucleotides . In DYE PRIMER SEQUENCING, the primer contains fluorescent dye – conjugated nucleotides, labeling the sequencing ladder at the 5 ′ ends of the chains. In DYE TERMINATOR SEQUENCING, the fluorescent dye molecules are covalently attached to the dideoxynucleotides , labeling the sequencing ladder at the 3 ′ ends of the chains. ddA ddA

30. Dye-terminator sequencing Dye-terminator sequencing  utilizes labelling of the chain terminator ddNTPs , which permits sequencing in a single reaction, rather than four reactions as in the labelled -primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which with different wavelengths  of fluorescence and emission.

31. Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing. The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects. Its limitations include dye effects due to differences in the incorporation of the dye- labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis  . This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs".

32. A C G T The fragments are distinguished by size and “color.” Dye Terminator Sequencing A distinct dye or “color” is used for each of the four ddNTP . Since the terminating nucleotides can be distinguished by color, all four reactions can be performed in a single tube. A T G T

33. Capillary G T C T G A Slab gel GA TC G A T C Dye Terminator Sequencing The DNA ladder is resolved in one gel lane or in a capillary. Capillary Slab gel 5 ′ A G T C T G Electropherogram The DNA ladder is read on an electropherogram .

34. 5 ′ A G T C T G 5 ′ A G( T/ A ) C T G 5 ′ A G A C T G T/T T/A A/A Automated Sequencing Dye primer or dye terminator sequencing on capillary instruments. Sequence analysis software provides analyzed sequence in text and electropherogram form. Peak patterns reflect mutations or sequence changes.

35. Other Improved Approaches and Automated DNA Sequencing Updated version of Sanger method Fluorescence detection with lasers Cycle sequencing Shotgun sequencing http://www.cmb.uab.edu/courses/lectures/scheirer2.pdf chsfpc5.chem.ncsu.edu/Poznan/ chem_bio/sld026.htm

36. opbs.okstate.edu/.../ sld015.htm

37. Alternative Sequencing Methods: Pyrosequencing Pyrosequencing  is a method of  DNA sequencing (determining the order of  nucleotides  in DNA) based on the " sequencing by synthesis " principle. The technique was developed by  Pål Nyrén  and his student  Mostafa Ronaghi  at the  Royal Institute of Technology  in Stockholm in 1996. It differs from  Sanger sequencing , relying on the detection of  pyrophosphate  release on nucleotide incorporation, rather than chain termination with  dideoxynucleotides .

38. Principle Pyrosequencing is based on the generation of light signal through release of pyrophosphate ( PPi ) on nucleotide addition . DNA n + dNTP  DNA n+1 + PP I PPi is used to generate ATP from adenosine phosphosulfate (APS). APS + PP I  ATP ATP and luciferase generate light by conversion of luciferin to oxyluciferin . Each nucleotide is added in turn. Only one of four will generate a light signal. The remaining nucleotides are removed enzymatically . The light signal is recorded on a pyrogram . DNA sequence: A T C A GG CC T Nucleotide added : A T C A G C T

39. Procedure "Sequencing by synthesis" involves taking a single strand of the DNA to be sequenced and then synthesizing its complementary strand enzymatically . Essentially, the method allows sequencing of a single strand of DNA  by synthesizing the complementary strand along it, one base pair at a time, and detected which base was actually added at each step. The Pyrosequencing method is based on detecting the activity of  DNA polymerase   with another  chemiluminescent   enzyme . The template DNA is immobile, and solutions of A, C, G, and T  nucleotides  are added and removed after the reaction, sequentially. Light is produced only when the nucleotide solution complements the first unpaired base of the template. The sequence of solutions which produce chemiluminescent signals allows the determination of the sequence of the template.

40. Alternative Sequencing Methods: Bisulfite Sequencing Bisulfite sequencing is used to detect methylation in DNA. Bisulfite deaminates cytosine , making uracil . Methylated cytosine is not changed by bisulfite treatment. The bisulfite -treated template is then sequenced.

41. The sequence of treated and untreated templates is compared. GTC Methylated sequence: GTC Me GGC Me GAT C TATC Me GTG C A … Treated sequence: Me GGC Me GAT U TATC Me GTG U A … DNA Sequence: (Untreated) reference: ...GT C GG C GAT C TAT C GTG C A… Treated sequence: ...GT C GG C GAT U TAT C GTG U A… This sequence indicates that these Cs are methylated. Alternative Sequencing Methods: Bisulfite Sequencing

42. Human Genome Project (HGP) HGP is a national effort to sequence and analyze the human genome which is a very complex system consisting of 50,000 to 10,0000 genes. These genes are located on 23 pairs of chromosome. The draft sequence was released in 2000. Some reasons for studying Human genome: Better medical practice High-quality diagnosis of diseases Understanding of evolution fully Improvement in biological research and forensic science Improvement in agriculture etc. The latest research on HGP are Pulsed electrophoresis Fluorescence microscopy 2D gel electrophoresis gtc double-stranded subclone inserts http://www.cem.msu.edu/~chem181h/projects/96/genome

43. opbs.okstate.edu/.../ sld015.htm

44. Summary Genetic information is stored in the order or sequence of nucleotides in DNA. Chain termination sequencing is the standard method for the determination of nucleotide sequence. Dideoxy-chain termination sequencing has been facilitated by the development of cycle sequencing and the use of fluorescent dye detection. Alternative methods are used for special applications, such as pyrosequencing (for resequencing and polymorphism detection) or bisulfite sequencing (to analyze methylated DNA).

45. Thanks Acknowledgement: All the material/presentations available online on the subject are duly acknowledged. Disclaimer: The author bear no responsibility with regard to the source and authenticity of the content. Questions???