1. DNA Sequencing

2. * Sequencing means finding the order of nucleotides on a piece of DNA. * Nucleotide order determines amino acid order, and by extension, protein structure and function (proteomics). *An alteration in a DNA sequence can lead to an altered or non functional protein, and hence to a harmful effect in a plant or animal

3. *Understanding a particular DNA sequence can shed light on a genetic condition and offer hope for the eventual development of treatment DNA. *Technology is also extended to environmental and agricultural.

4. Methods of sequencing 1-Sanger dideoxy (primer extension/chain-termination) method: most popular protocol for sequencing, very adaptable, scalable to large sequencing projects 2-Maxam-Gilbert chemical cleavage method: DNA is labelled and then chemically cleaved in a sequence- dependent manner. This method is not easily scaled and is rather tedious. *there are two main methods of DNA sequencing: *Modern sequencing equipment uses the principles of the Sanger technique.

5. History of Sequencing ”Sanger Sequencing” developed by Fred Sanger et al in the mid 1970’s Uses dideoxynucleotides for ”chain termination”, generating fragments of different lengths ending in ddATP, ddGTP, ddCTP or ddTTP

6. The Sanger Technique Principle : -The Sanger Technique uses dideoxynucleotides (dideoxyadenine, dideoxyguanine, etc) These are molecules that resemble normal nucleotides but lack the normal -OH group. -Because they lack the -OH (which allows nucleotides to join a growing DNA strand), replication stops. -Normally, this would be where another phosphate is attached, but with no -OH group, a bond can not form and replication stops.

7. Requirements for Sanger Method DNA to be sequenced must be in single strand form. The region to be sequenced must be 3’ flanked by known sequence. Reagents needed are: – A primer complementary to the known region to start and direct chain synthesis. (15-30 nucleotides in length) – DNA polymerase. – 4 deoxynucleotide triphosphates (dNTPs). – 4 dideoxynucleotide triphosphates (ddNTPs) ( small proportion ).

8. Dideoxynucleotides dATPddATP The 3’ hydroxyl has been changed to a hydrogen in ddNTP’s, which terminates a DNA chain because a phosphodiester bond cannot form at this 3’ location

9. DNA polymerase catalyzed nucleophilic attack of the 3’-OH on a phospho-anhydride ** Since the 3’ –OH is changed to a –H in ddNTPs, it is unable to form a phosphodiester bond by nucleophilic attack on the phosphate, and it will cause a termination in the DNA chain Mechanism of DNA polymerization : : 5’ 3’ 5’ 3’

10. Sanger Method Partial copies of DNA fragments made with DNA polymerase Collection of DNA fragments that terminate with A,C,G or T using ddNTP Separate by gel electrophoresis Read DNA sequence

11. *The template DNA pieces are replicated, incorporating normal nucleotides, but occasionally and at random dideoxy (DD) nucleotides are taken up. *This stops replication on that piece of DNA. *The result is a mix of DNA lengths, each ending with a particular labeled DDnucleotide. *Because the different lengths ‘travel’ at different rates during electrophoresis, their order can be determined.

12. Sanger dideoxy sequencing: basic method 5’ 3’ 5’ 3’ TTTT ddA ddATP in the reaction: anywhere there’s a T in the template strand, occasionally a ddA will be added to the growing strand

13. Sequencing of DNA by the Sanger method

14. CCGTAC 3’ 5’ 3’ primer dNTP ddATP GGCA ddTTP GGCAT ddCTP GGCG ddGTP G GGCATG A T C G 5’ 3’ the sequence is complementary to the original strand

15. *Originally four separate sets of DNA, primer and a single different DD nucleotide were produced and run on a gel. *Modern technology allows all the DNA, primers, etc to be mixed and the fluorescent labeled DDnucleotide ‘ends’ of different lengths can be ‘read’ by a laser. *Additionally, the gel slab has been replaced by polymer filled capillary tubes in modern equipment.

16. Gel Separation The reaction mixtures are separated on a denaturing polyacrylamide gel. – Denaturing to prevent the DNA from folding up on itself while it travels through. – Polyacrylamide to separate the strands which differ in length by only one nucleotide. Each band corresponds to a sequence of DNA which was terminated by a particular ddNTP. This ddNTP is identified by lane in the radioactive method and by color in the fluorescent method. The lowest band on the gel is the shortest. The shorter the strand, the earlier in the synthetic reaction the ddNTP was incorporated. The lowest band on the gel is at the 5’ end of our synthesized strand and is complementary to the 3’ end of our unknown fragment.

17. Sequencing Visualization Methods Two forms of labeling: – Radioactive Primer labeled ( 32 P or 33 P) dNTP labeled ( 35 S or 32 P) – Nonradioactive ( Fluorescence ) *Primer labeled *ddNTP labeled - ddNTPs chemically synthesized to contain fluorescence. - Each ddNTP fluoresces at a different wavelength allowing identification.

18. Gel Visualization Radioactive method which requires four gel lanes, one for each reaction vessel. – Readout is done by hand or with a densitometric scanner. Nonradioactive fluorescence sequencing requires only one gel lane because each nucleotide has a distinct color. – The readout process is done by laser scanner and recorded by computer.

20. Nonradioactive ddGTP ddATP ddTTP ddCTP Radioactive Sequence of unknown fragment vs. Gel Electrophoresis and Readout of Reaction Products: Sequence of unknown fragment Shortest synthesized band = 5’ end of synthesized strand Longest synthesized band = 3’ end of synthesized strand

21. Manual vs. Automatic Sequencing Comparison listManualAutomatic DNA labelingRadioisotopeFluorescence dye Signal detectFilmPhotomultiplier Computer Sample4 lanes / 1 sequencing sample 1 lane / 1 sequencing sample Read length (average) 250 ∼ 500 bp400 ∼ 1000 bp

23. Sample Output 1 lane

24. An automated sequencer The output

25. Radioactive Primer Labeled Sequencing 4. dNTP’s ( dATP, dGTP, dCTP, and dTTP) ddGTPddATPddCTPddTTP 6. One type of ddNTP per reaction Remember each reaction has many molecules each one incorporating its respective ddNTP and stopping at a different length. 7. DNA polymerase 3. Complementary primer, 5’end-labeled with 32 P or 33 P 5’ 2. with region of known sequence 3’ 1. Unknown fragment 5’ Reaction 2Reaction 1Reaction 3Reaction 4 5. Four separate reactions 5’ 3’ 5’ 8. ddNTP incorporation - stops chain synthesis 3’

26. Radioactive Deoxynucleotide Labeled Sequencing ddGTPddATPddCTPddTTP 6. One type of ddNTP per reaction 7. DNA Polymerase2. with region of known sequence 3’ 8. ddNTP incorporation - stops chain synthesis 3’ 3. Complementary primer 5’ 1. Unknown fragment 5’ Reaction 1Reaction 2Reaction 3Reaction 4 5. Four separate reactions 3’ 5’ 4. dNTP’s 35 S labeled dATP or dCTP

27. Fluorescent Primer Labeled Sequencing 5. dNTP’s (dATP, dGTP, dCTP, and dTTP) ddGTPddATPddCTPddTTP 6. One type of ddNTP per reaction What’s the big advantage here? 7. DNA Polymerase 1. Unknown fragment 5’ 4. Fluorescent labeled primer. Different fluorescent dye per reaction 5’ 8. ddNTP incorporation - stops chain synthesis 3’ 2. with region of known sequence 3’ 5’ Reaction 1Reaction 2Reaction 3Reaction 4 3. Four separate reactions 5’ 3’

28. Fluorescent Dideoxynucleotide Labeled Sequencing 4. dNTP’s (dATP, dGTP, dCTP, and dTTP) ddGTP ddCTP ddTTP ddATP 5. Fluorescent labeled ddNTP’s. Each labeled with a different fluorescent dye Now we run our products on gel 6. DNA Polymerase 3. Complementary primer 5’ Don’t forget that this and the all the previous reaction vessels have millions of our unknown fragment. Why do you think we’re only showing 4 representatives? One reaction vessel 1. Here we have one reaction vessel, with four copies of our Unknown fragment. 5’ 2. A region of known sequence 3’ 7. Again ddNTP incorporation stops chain synthesis 3’

29. Strategy I – Four different reaction mixtures are set up – Primers covalently bonded to fluorescing dye at the 5’-terminus – Reaction products are separated by gel electrophoresis in parallel lanes – Laser induced fluorescence (LIF) is detected Strategy II – Primers in each reaction mixture are labeled with different fluorescent dyes – Reaction mixtures are mixed at the end of the reaction and separated in a single lane by gel electrophoresis – The terminal base of each fragment is identified by the fluorescence of the dye on the associated primer Commercial San ger

30. Strategy III – A single vessel used – Each of the ddNTPs is covalently bonded to a different fluorescent dye – The products are separated in single lane – The terminal base is identified by the characteristic fluorescence of the dye attached to the terminator Commercial Sanger

31. Chemical Degradation Method Maxam-Gilbert method – 5’ end marked with radioactive label. – Chemical reaction randomly cleaves strands of DNA at specific locations: G, A (some G), T (some C), C. – Results in strands of varying lengths. – Strands separated out with electrophoresis. – Gels read with radioautography.

32. Labeled DNA is treated with a reagent that cleaves DNA at a particular type of nucleotide. Hydrazine cleaves DNA before every C-nucleotide at 1.5 M NaCl. Reaction must have low yield so as to obtain random distribution of different length due to cuts at all the sites. Labeled fragments are separated by SDS-PAGE. Chemical Cleavage/ Maxam-Gilbert Method)

33. Aliquot 1 Cleavage at only G – DNA treated with Dimethyl sulfate (DMS) – Methylation of G residues at the N7 position – the glycoside bond of the methylated G residue is hydrolyzed and the G residue is eliminated. – Piperidine is added which reacts with hydrolyzed sugar residue, cleavage of the backbone results Aliquot 2 cleavage at G and A – Use acid instead of DMS – Position of A revealed Aliquot 3: cleavage at C and T – Treat with hydrazine, then piperidine Aliquot 4: cleavage at C only – Treat with hydrazine in the presence of 1.5 M NaCl – Position of T revealed Reagents for cleaving DNA

35. Labeled DNA to be sequenced – 32P-ACCTGACATCG Cleavage products – 32P-ACCTGACAT – 32P-ACCTGA – 32P-AC – 32P-A Example of cleavage before C

38. Analysis of sequencing products: Polyacrylamide gel electrophoresis--good resolution of fragments differing by a single dNTP –Capillary gels: require only a tiny amount of sample to be loaded, run much faster than slab gels, best for high throughput sequencing

39. Comparison Sanger Method – Enzymatic – Requires DNA synthesis – Termination of chain elongation Maxam Gilbert Method – Chemical – Requires DNA – Requires long stretches of DNA – Breaks DNA at different nucleotides