PCR, genome sequencing: Science House, Apr 17, 2004
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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) 1. chain termination sequencing single-stranded, denatured DNA ACTTGTGCGATG 1. chain termination sequencing single-stranded, denatured DNA ACTTGTGCGATG reaction buffer, DNA polymerase, dNTPs, ddNTPs, primer ATCG ATCG TAC 1. chain termination sequencing single-stranded, denatured DNA ACTTGTGCGATG reaction buffer, DNA polymerase, dNTPs, ddNTPs, primer ATCG ATCG randomly incorporated, ddNTPs stop the reaction, resulting in a nested set of DNA fragments TGAACACGCTAC GAACACGCTAC AACACGCTAC ACACGCTAC CACGCTAC ACGCTAC CGCTAC GCTAC CTAC TAC 1. chain termination sequencing single-stranded, denatured DNA ACTTGTGCGATG reaction buffer, DNA polymerase, dNTPs, ddNTPs, primer ATCG ATCG randomly incorporated, ddNTPs stop the reaction, resulting in a nested set of DNA fragments TGAACACGCTAC GAACACGCTAC AACACGCTAC ACACGCTAC CACGCTAC ACGCTAC CGCTAC GCTAC CTAC DNA fragments are separated by electrophoresis TAC 2. polymerase chain reaction (PCR) iterative process, consists of 3 steps: 1.denaturation of the template DNA by heat 2. polymerase chain reaction (PCR) 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) 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) 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 30th cycle: 230 x 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 4. large scale sequencing (shotgun sequencing) 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) 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) 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) 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 (hierarchical sequencing) 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) 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) 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) 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 hierarchical sequencing vs. shotgun sequencing + filling gaps, resequencing uncertain regions is easier + physical map construction is not necessary + distribute clones to different labs + cost effective and fast + good for small genomes - constructing the physical map is expensive and time-consuming - filling gaps and keeping track of sequenced plasmids is more difficult - computationally more expensive hierarchical sequencing vs. shotgun sequencing + filling gaps, resequencing uncertain regions is easier + physical map construction is not necessary + distribute clones to different labs + cost effective and fast + good for small genomes - constructing the physical map is expensive and time-consuming - filling gaps and keeping track of sequenced plasmids is more difficult - computationally more expensive more info on PCR:
