DNA sequencing Flashcards
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 _______
specific primer. DNA polymerase Base-specific termination - one dNTP - ddNTPs - polyacrylamide gels - ssDNA differeing in length by one nucleotide - autoradiographs
Sanger primers
Specific primers: ____________
Universal Primers: __________
Similar to requirements for PCR primers
Anneal to vector sequences flanking the “Multiple Cloning Site
DNA templates in Sanger Sequencing
Best templates for classical Sanger Sequencing: _______
ssDNA isolated from recombinant M13 bacteriophages
DNA templates in Sanger Sequencing (current methods)
dsDNA (denatured by heating) involving Plasmid DNA and PCR-amplified DNA
The ratio of dNTP::ddNTP in Sanger Sequencing determines ____________ and also enables flexibility in __________
This is due to an assumption that _________
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
With a higher ratio of dNTP::ddNTP
The likelihood of extending the chain increases
32P ( α-32P[dNTP]) radiolabelling has _____ activity
_____ half life
______ on gel
_____ concerns
high
short
more diffuse bands
safety
35S (or 33p) radiolabelling has
_________ activity
_______ half life
______ on sequencing gels
Lower
Longer
Better resolution
DNA sequencing gels are
_______ ___________ gels
The are thin to _______
Urea is used as a ______ to keep DNA in _______
Running at higher temperatures will __________
denaturing polyacrylamide gels
improve resolution
denaturant, single-stranded form
reduce compression
Some major improvements for the sanger method are (5)
Fluorescent labelling Automation Cycling sequencing using thermostable DNA polymerase Capillary electrophoresis Software for improved base calling
Fluorescent labelling use in
Signals can be _______ and ______________ with _______
modified polymerases used to solve _________
Automated DNA sequencers
detected, processed continuously, CCD cameras (photomultiplier tubes
problems with incorporation efficiency
Important properties for DNA polymerases in sequencing
High _______
__________
Do not have _________
having similar efficiencies in __________
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
original taq polymerase not optimal for sequencing because it has ________ and _______, although it is _______ which is good
5’->3’ exonuclease activity and a preference for dNTPs over ddNTPs or fluorescent analogs (factor of ~ 10^3)
thermostable
thermostability in sequencing reactions is desired because
sequencing reactions at elevated temperatures reduce templates rich in secondary structures
engineered taq
3’->5’ exonuclease activity is ___________
_________ reduces preference for dNTPs over ddNTPs or fluorescent analogs
eliminated by point mutation or deletion
F667Y
Illumina
Sequencing by synthesis with fluorescently labelled reversible terminators
Life technologies (ION Torrent)
Semiconductor sensor arrays detect protons released by nucleotide addition
Roche(454)
Pyrosequencing of template-laden beads prepared by emulsion PCR
Pyrosequencing steps
- 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
On an instrument the picotiter plate acts as a
flow cell into which each pure nucleotide is added step by step
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 \_\_\_\_\_\_\_\_\_\_\_
nebulization
cloning, colony picking
adapters
avidin/biotin purification
In emulsion PCR
____________ to an excess of DNA capture beads
Emulsify beads and ________ in ___________(MR)
________ occurs inside the MR
Break the MR and ________ for __________
Anneal ssDNA
PCR reagents, water-in-oil microreactors
Clonal amplification
Enrich, DNA-positive beads
Pyrosequencing Sequencing Well diameter average of \_\_\_\_\_\_ 400,000 \_\_\_\_\_\_ obtained in \_\_\_\_\_\_\_\_\_ A single \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ is deposited per well
44 µM
reads, parrallel
cloned amplified DNA read in deposited per well
