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: