sequencing-related topics
DESCRIPTION
SEQUENCING-related topics. chain-termination sequencing the polymerase chain reaction (PCR) cycle sequencing large scale sequencing. stefanie . hartmann @ unc . edu (postdoc in Todd Vision’s lab). 1. chain termination sequencing. single-stranded, denatured DNA. - PowerPoint PPT PresentationTRANSCRIPT
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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)
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1. chain termination sequencing
single-stranded, denatured DNA A C T T G T G C G A T G
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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 CA T C G A T C G
1. chain termination sequencing
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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 CA C A C G C T A C
C A C G C T A CA 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 A T C G
1. chain termination sequencing
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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 CA C A C G C T A C
C A C G C T A CA 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 A T C G
1. chain termination sequencing
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2. polymerase chain reaction (PCR)
iterative process, consists of 3 steps:
1.denaturation of the template DNA by heat
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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)
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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)
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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
2. polymerase chain reaction (PCR)
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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
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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)
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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)
![Page 13: SEQUENCING-related topics](https://reader036.vdocument.in/reader036/viewer/2022062500/5681522b550346895dc07438/html5/thumbnails/13.jpg)
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)
![Page 14: SEQUENCING-related topics](https://reader036.vdocument.in/reader036/viewer/2022062500/5681522b550346895dc07438/html5/thumbnails/14.jpg)
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)
![Page 15: SEQUENCING-related topics](https://reader036.vdocument.in/reader036/viewer/2022062500/5681522b550346895dc07438/html5/thumbnails/15.jpg)
WHOLE GENOMEbreak into random fragments
FRAGMENTS
clone into BAC vectors,
map fragments
PHYSICAL MAP
fragment and subclone inserts intoplasmid 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)
![Page 16: SEQUENCING-related topics](https://reader036.vdocument.in/reader036/viewer/2022062500/5681522b550346895dc07438/html5/thumbnails/16.jpg)
WHOLE GENOMEbreak into random fragments
FRAGMENTS
clone into BAC vectors,
map fragments
PHYSICAL MAP
fragment and subclone inserts intoplasmid 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)
![Page 17: SEQUENCING-related topics](https://reader036.vdocument.in/reader036/viewer/2022062500/5681522b550346895dc07438/html5/thumbnails/17.jpg)
WHOLE GENOMEbreak into random fragments
FRAGMENTS
clone into BAC vectors,
map fragments
PHYSICAL MAP
fragment and subclone inserts intoplasmid 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)
![Page 18: SEQUENCING-related topics](https://reader036.vdocument.in/reader036/viewer/2022062500/5681522b550346895dc07438/html5/thumbnails/18.jpg)
WHOLE GENOMEbreak into random fragments
FRAGMENTS
clone into BAC vectors,
map fragments
PHYSICAL MAP
fragment and subclone inserts intoplasmid 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)
![Page 19: SEQUENCING-related topics](https://reader036.vdocument.in/reader036/viewer/2022062500/5681522b550346895dc07438/html5/thumbnails/19.jpg)
+ 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
![Page 20: SEQUENCING-related topics](https://reader036.vdocument.in/reader036/viewer/2022062500/5681522b550346895dc07438/html5/thumbnails/20.jpg)
+ 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
![Page 21: SEQUENCING-related topics](https://reader036.vdocument.in/reader036/viewer/2022062500/5681522b550346895dc07438/html5/thumbnails/21.jpg)
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more info on PCR: