SEQUENCING-related topics

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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 _at_ unc.edu (postdoc in Todd
Visions 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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1. chain termination sequencing
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 A T C G
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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
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 A T C G
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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
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 A T C G
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2. polymerase chain reaction (PCR)

• iterative process, consists of 3 steps
• denaturation of the template DNA by heat

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2. polymerase chain reaction (PCR)

• iterative process, consists of 3 steps
• denaturation of the template DNA by heat
• annealing of the oligonucleotide primers to the
  single-stranded target sequence

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2. polymerase chain reaction (PCR)

• iterative process, consists of 3 steps
• denaturation of the template DNA by heat
• annealing of the oligonucleotide primers to the
  single-stranded target sequence
• extension of the annealed primers by a
  thermostable DNA polymerase

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2. polymerase chain reaction (PCR)

• iterative process, consists of 3 steps
• denaturation of the template DNA by heat
• annealing of the oligonucleotide primers to the
  single-stranded target sequence
• 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

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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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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 reseq
uence regions between contigs if necessary WHOLE
GENOME SEQUENCE
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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 reseq
uence regions between contigs if necessary WHOLE
GENOME SEQUENCE
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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 reseq
uence regions between contigs if necessary WHOLE
GENOME SEQUENCE
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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 reseq
uence regions between contigs if necessary WHOLE
GENOME SEQUENCE
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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
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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
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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
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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
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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
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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
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more info on PCR