DNA sequencing

Question 1

Important features in Sanger sequencing
Use of a ___________ for extension by _________
_____ _____ ____ (4 reactions)
- limit the concentration of ______
- introduction of ______ into the reaction
Use of ___________ to discriminate between ______
Detection of synthesized ssDNAs by _______

Answer 1

specific primer. DNA polymerase
Base-specific termination
- one dNTP
- ddNTPs
- polyacrylamide gels
- ssDNA differeing in length by one nucleotide
- autoradiographs

Question 2

Sanger primers
Specific primers: ____________
Universal Primers: __________

Answer 2

Similar to requirements for PCR primers

Anneal to vector sequences flanking the “Multiple Cloning Site

Question 3

DNA templates in Sanger Sequencing

Best templates for classical Sanger Sequencing: _______

Answer 3

ssDNA isolated from recombinant M13 bacteriophages

Question 4

DNA templates in Sanger Sequencing (current methods)

Answer 4

dsDNA (denatured by heating) involving Plasmid DNA and PCR-amplified DNA

Question 5

The ratio of dNTP::ddNTP in Sanger Sequencing determines ____________ and also enables flexibility in __________
This is due to an assumption that _________

Answer 5

frequency of incorporation of ddNTP, and hence termination
Determining sequences closer or further away from the DNA binding site
The rate of usage of dNTPs and ddNTPs by the DNA polymerase is the same

Question 6

With a higher ratio of dNTP::ddNTP

Answer 6

The likelihood of extending the chain increases

Question 7

32P ( α-32P[dNTP]) radiolabelling has _____ activity
_____ half life
______ on gel
_____ concerns

Answer 7

high
short
more diffuse bands
safety

Question 8

35S (or 33p) radiolabelling has
_________ activity
_______ half life
______ on sequencing gels

Answer 8

Lower
Longer
Better resolution

Question 9

DNA sequencing gels are
_______ ___________ gels
The are thin to _______
Urea is used as a ______ to keep DNA in _______
Running at higher temperatures will __________

Answer 9

denaturing polyacrylamide gels
improve resolution
denaturant, single-stranded form
reduce compression

Question 10

Some major improvements for the sanger method are (5)

Answer 10

Fluorescent labelling
Automation
Cycling sequencing using thermostable DNA polymerase
Capillary electrophoresis
Software for improved base calling

Question 11

Fluorescent labelling use in
Signals can be _______ and ______________ with _______
modified polymerases used to solve _________

Answer 11

Automated DNA sequencers
detected, processed continuously, CCD cameras (photomultiplier tubes
problems with incorporation efficiency

Question 12

Important properties for DNA polymerases in sequencing
High _______
__________
Do not have _________
having similar efficiencies in __________

Answer 12

processivity (rate of DNA polymerization)
thermostability (more important in cycle sequencing)
exonuclease activities (5’->3’ removes primers, 3’->5’ discriminates against ddNTPs and pauses at secondary structures)
incorporation of ddNTP primers

Question 13

original taq polymerase not optimal for sequencing because it has ________ and _______, although it is _______ which is good

Answer 13

5’->3’ exonuclease activity and a preference for dNTPs over ddNTPs or fluorescent analogs (factor of ~ 10^3)
thermostable

Question 14

thermostability in sequencing reactions is desired because

Answer 14

sequencing reactions at elevated temperatures reduce templates rich in secondary structures

Question 15

engineered taq
3’->5’ exonuclease activity is ___________
_________ reduces preference for dNTPs over ddNTPs or fluorescent analogs

Answer 15

eliminated by point mutation or deletion

F667Y

Question 16

Illumina

Answer 16

Sequencing by synthesis with fluorescently labelled reversible terminators

Question 17

Life technologies (ION Torrent)

Answer 17

Semiconductor sensor arrays detect protons released by nucleotide addition

Question 18

Roche(454)

Answer 18

Pyrosequencing of template-laden beads prepared by emulsion PCR

Question 19

Pyrosequencing steps

Answer 19

• Prepare libraries of DNA fragments linked to adapters and isolate ss fragments with A/B adapters
• DNA fragments (ss) are mixed with agarose beads carrying oligonucleotides complementary to the adapters under conditions to have one fragment/bead
• Clonal amplification
• each bead placed in a tiny well in picotiter plate, and DNA it carries is used as template for sequencing

Question 20

On an instrument the picotiter plate acts as a

Answer 20

flow cell into which each pure nucleotide is added step by step

Question 21

In DNA library preparation
The genome is fragmented by \_\_\_\_\_\_\_
There is no \_\_\_\_\_\_ and no \_\_\_\_\_\_
The ssDNA library is created with \_\_\_\_\_\_\_\_\_\_
A/B fragments selected using \_\_\_\_\_\_\_\_\_\_\_

Answer 21

nebulization
cloning, colony picking
adapters
avidin/biotin purification

Question 22

In emulsion PCR
____________ to an excess of DNA capture beads
Emulsify beads and ________ in ___________(MR)
________ occurs inside the MR
Break the MR and ________ for __________

Answer 22

Anneal ssDNA
PCR reagents, water-in-oil microreactors
Clonal amplification
Enrich, DNA-positive beads

Question 23

Pyrosequencing
Sequencing
Well diameter average of \_\_\_\_\_\_
400,000 \_\_\_\_\_\_ obtained in \_\_\_\_\_\_\_\_\_
A single \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ is deposited per well

Answer 23

44 µM
reads, parrallel
cloned amplified DNA read in deposited per well