1. SEQUENCING-related topics 1. chain-termination sequencing 2. the polymerase chain reaction (PCR) 3. cycle sequencing 4. large scale sequencing stefanie.hartmann @ unc.edu (postdoc in Todd Vision’s lab)

2. 1. chain termination sequencing single-stranded, denatured DNAA C T T G T G C G A T G

3. single-stranded, denatured DNA reaction buffer, DNA polymerase, dNTPs, ddNTPs, primer A C T T G T G C G A T G T A C A T C G 1. chain termination sequencing

4. single-stranded, denatured DNA reaction buffer, DNA polymerase, dNTPs, ddNTPs, primer randomly incorporated, ddNTPs stop the reaction, resulting in a nested set of DNA fragments A C T T G T G C G A T G T A C T G A A C A C G C T A C G A A C A C G C T A C A A C A C G C T A C A C A C G C T A C C A C G C T A C A C G C T A C C G C T A C G C T A C C T A C A T C G 1. chain termination sequencing

5. single-stranded, denatured DNA reaction buffer, DNA polymerase, dNTPs, ddNTPs, primer randomly incorporated, ddNTPs stop the reaction, resulting in a nested set of DNA fragments DNA fragments are separated by electrophoresis A C T T G T G C G A T G T A C T G A A C A C G C T A C G A A C A C G C T A C A A C A C G C T A C A C A C G C T A C C A C G C T A C A C G C T A C C G C T A C G C T A C C T A C A T C G 1. chain termination sequencing

6. 2. polymerase chain reaction (PCR) iterative process, consists of 3 steps: 1.denaturation of the template DNA by heat

7. iterative process, consists of 3 steps: 1.denaturation of the template DNA by heat 2. annealing of the oligonucleotide primers to the single-stranded target sequence 2. polymerase chain reaction (PCR)

8. iterative process, consists of 3 steps: 1.denaturation of the template DNA by heat 2. annealing of the oligonucleotide primers to the single-stranded target sequence 3. extension of the annealed primers by a thermostable DNA polymerase 2. polymerase chain reaction (PCR)

9. iterative process, consists of 3 steps: 1.denaturation of the template DNA by heat 2. annealing of the oligonucleotide primers to the single-stranded target sequence 3. extension of the annealed primers by a thermostable DNA polymerase repeat for 30-40 cycles; each cycle doubles the amount of DNA synthesized in the previous cycle - after 30 th cycle: 2 30 x 2. polymerase chain reaction (PCR)

10. 3. (thermal) cycle sequencing (linear amplification DNA sequencing) contains sequencing reaction mixture of buffer, template, DNA polymerase, primer, dNTP, ddNTP consists, like a standard PCR, of cycles of denaturation, annealing, and extension BUT: uses only one primer to linearly amplify the extension products

11. WHOLE GENOME break into random fragments FRAGMENTS clone into plasmid vectors CLONE LIBRARY sequence fragments without knowledge of their chromosomal location THOUSANDS OR MILLIONS OF SHORT SEQUENCES use a computer to assemble the entire sequence from the overlaps found CONTIGS resequence regions between contigs if necessary WHOLE GENOME SEQUENCE 4. large scale sequencing (shotgun sequencing)

12. WHOLE GENOME break into random fragments FRAGMENTS clone into plasmid vectors CLONE LIBRARY sequence fragments without knowledge of their chromosomal location THOUSANDS OR MILLIONS OF SHORT SEQUENCES use a computer to assemble the entire sequence from the overlaps found CONTIGS resequence regions between contigs if necessary WHOLE GENOME SEQUENCE 4. large scale sequencing (shotgun sequencing)

13. WHOLE GENOME break into random fragments FRAGMENTS clone into plasmid vectors CLONE LIBRARY sequence fragments without knowledge of their chromosomal location THOUSANDS OR MILLIONS OF SHORT SEQUENCES use a computer to assemble the entire sequence from the overlaps found CONTIGS resequence regions between contigs if necessary WHOLE GENOME SEQUENCE 4. large scale sequencing (shotgun sequencing)

14. WHOLE GENOME break into random fragments FRAGMENTS clone into plasmid vectors CLONE LIBRARY sequence fragments without knowledge of their chromosomal location THOUSANDS OR MILLIONS OF SHORT SEQUENCES use a computer to assemble the entire sequence from the overlaps found CONTIGS resequence regions between contigs if necessary WHOLE GENOME SEQUENCE 4. large scale sequencing (shotgun sequencing)

15. WHOLE GENOME break into random fragments FRAGMENTS clone into BAC vectors, map fragments PHYSICAL MAP fragment and subclone inserts into plasmid vectors CLONE LIBRARY sequence the clones SHORT SEQUENCES use a computer to assemble the entire sequence from the overlaps found CONTIGS resequence regions between contigs if necessary WHOLE GENOME SEQUENCE 4. large scale sequencing (hierarchical sequencing)

16. WHOLE GENOME break into random fragments FRAGMENTS clone into BAC vectors, map fragments PHYSICAL MAP fragment and subclone inserts into plasmid vectors CLONE LIBRARY sequence the clones SHORT SEQUENCES use a computer to assemble the entire sequence from the overlaps found CONTIGS resequence regions between contigs if necessary WHOLE GENOME SEQUENCE 4. large scale sequencing (hierarchical sequencing)

17. WHOLE GENOME break into random fragments FRAGMENTS clone into BAC vectors, map fragments PHYSICAL MAP fragment and subclone inserts into plasmid vectors CLONE LIBRARY sequence the clones SHORT SEQUENCES use a computer to assemble the entire sequence from the overlaps found CONTIGS resequence regions between contigs if necessary WHOLE GENOME SEQUENCE 4. large scale sequencing (hierarchical sequencing)

18. WHOLE GENOME break into random fragments FRAGMENTS clone into BAC vectors, map fragments PHYSICAL MAP fragment and subclone inserts into plasmid vectors CLONE LIBRARY sequence the clones SHORT SEQUENCES use a computer to assemble the entire sequence from the overlaps found CONTIGS resequence regions between contigs if necessary WHOLE GENOME SEQUENCE 4. large scale sequencing (hierarchical sequencing)

19. + filling gaps, resequencing uncertain regions is easier + distribute clones to different labs - constructing the physical map is expensive and time-consuming + physical map construction is not necessary + cost effective and fast + good for small genomes - filling gaps and keeping track of sequenced plasmids is more difficult - computationally more expensive hierarchical sequencing vs. shotgun sequencing

20. + filling gaps, resequencing uncertain regions is easier + distribute clones to different labs - constructing the physical map is expensive and time-consuming + physical map construction is not necessary + cost effective and fast + good for small genomes - filling gaps and keeping track of sequenced plasmids is more difficult - computationally more expensive hierarchical sequencing vs. shotgun sequencing

23. more info on PCR: