2. Sanger-Coulson Dideoxynucleotide Sequencing Kwamina Bentsi-Barnes Deisy Mendoza Jennifer Aoki Lecture 10/30/00 Best printed in color for clarity

3. Topics of Discussion Review of last lecture. Sequencing. Requirements for Sanger-Coulson sequencing. Dideoxynucleotides. –Mechanism of DNA polymerization. Sequencing visualization methods. –Radioactive primer labeled sequencing. –Radioactive dNTP labeled sequencing. –Fluorescent primer labeled sequencing. –Fluorescent ddNTP labeled sequencing. Gel separation. Gel visualization. –Gel electrophoresis and readout Relative template quantities for sequencing.

4. Review of Last Lecture on 10/25/00 Southern blot cDNA probe Good vectors –pGEM -3zf(+/-) Lambda –Replicate independently –Fairly small –cos-sites won’t package, endonucleases recognize those sites –cos site is required for package either in vivo or in vitro Super Cos –A lot smaller lambda –Can replicate individually as a plasmid

5. Sequencing Sequencing is the process by which you determine the exact order of the nucleotides in a given region of DNA. Dideoxynucleotide sequencing is done through complementary chain synthesis and early termination. The synthesized chains are visualized by methods using: –Radioactive labels. –Nonradioactive labels.

6. Requirements for Sanger- Coulson Sequencing 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 direct chain synthesis. –DNA polymerase. –4 deoxynucleotide triphosphates (dNTPs). –4 dideoxynucleotide triphosphates (ddNTPs).

7. 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 Here is an example comparing dATP and ddATP :

8. 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’

9. Sequencing Visualization Methods Two forms of labeling: –Radioactive Primer labeled ( 32 P or 33 P) dNTP labeled ( 35 S) –Nonradioactive Primer labeled ddNTP labeled (big dye terminator)

10. 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’

11. Radioactive Deoxynucleotide Labeled Sequencing ddGTPddATPddCTPddTTP 6. One type of ddNTP per reaction What is different about this method? (hint: look at the colors) 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

12. 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’

13. 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’

14. 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.

15. 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.

16. 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

17. Most Double stranded plasmid (must denature) & standard sequencing reaction Each molecule of template used only once Less Single stranded construct such as phagemid or M13 & standard sequencing Each molecule of template used only once Least Either double stranded or single stranded construct types or fragments & cycle sequencing Each molecule is used as template up to 30 times Relative Template Quantities needed for Sequencing