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 A C T T G T G C G A T G
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
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
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
2. polymerase chain reaction (PCR)
iterative process, consists of 3 steps:
1.denaturation of the template DNA by heat
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
2. polymerase chain reaction (PCR)
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
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 (shotgun sequencing)
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)
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)
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)
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)
+ 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
+ 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
more info on PCR:
