Sequencing Flashcards
what is the ideal ratio of dNTP:ddNTP?
5:1
what types of radiolabelling can be used for PCR?
• 32P(α-32P[dNTP]) – High activity, safety concerns – Short half-life – More diffuse bands on gel Radiolabeling • 35S(or33P) – Lower activity – Longer half-life – Better resolution on sequencing gels
describe DNA sequencing gels
• Denaturing polyacrylamide gels
• Thin(0.2-0.5mmthick)toimproveresolution
• Urea is used as a denaturant to keep DNA in single-stranded form
• Could be run at higher temperatures to reduce “compression”
–long and thin
fluorescent labelling of ddNTPs–>each ddNTP has a different colour, move past detector–>get colour, figure out base info
ya
important points about DNA pols in sanger seq?
Do not have exonuclease activities
– 5’ - 3’ exonuclease activity: removal of primers
– 3’ - 5’ exonuclease activity: discrimination against ddNTPs and pausing at secondary structures
Describe illumina library prep
DNA fragments, cap with nts; phosphorylate ends; add overhanging A–>ligate to adapters
illumina–like bridge PCR, but without the bridging
ya
describe illumina seq
one nt put on at a time; each base has diff coloured fluorophore and a reversible block–allows to add one at a time
describe ion torrent gene seq
personal genome machine–200 bp long fragments connected to a single bead, as in emulsion PCR; when a base is added, a proton is released–detect change in pH; if no base is added then no pH change will be detected–do one round of one base; wash away; do another base; wash out, anther bae; etc; if two T’s are close to each other and dNTTs are in the mix, then you will get double the pH change
what are teh 3rd gen seq methods?
- Sequencing by hybridization/ligation e.g.
Complete Genomics - Single molecule sequencing by synthesis e.g. Pacific Biosciences (PacBio)
- Nanopore sequencing
Oxford Nanopore Technologies
- Nucleotides are driven through nanopores
- Differences in conductance can identify nucleotides
what is complete genomics?
break DNA into 400-500 pieces by sonication; cut (linearize) ad adapter 1, recirc; cut 13 bp to the right of ad 1, add ad 2; recirc; cut 13 bp to the left of AD 1, recirc; cut 26 bp to both right and left and add ad 4;
nanoball–insert 4 adapter fragments into DNA circ (400-500bp);then take phi29 pol and do long rolling replication–?create a long ssDNA–>forms to DNA nanoball; anchor onto silicone chip
–use a primer labelled to a one of four probes in a set–study slide 25–hybridize a different probe of one of four probes, each ontaining a different nt at a base–>the one which matches will stick on at high temps; the one that does match will be ligated to probe that is bound to anchor/adaptor; tell which base it is in that position bc probe is labelled with fluorophore corresponding to that base
what is SMRT (pacific biosciences)
single-molecule real time seq–anchor phi29 to bottom of well via biotin-streptavidin binding; feed template; dNTPs are labelled with several fluorphores to increase fluorescence;
- during synthesis, the nucleotide being incorporated is held by the polymerase, resulting in longer signal
- free diffusing nucleotide passing the illuminated area faster and generates much shorter signal
describe nanopore seq (TGS)
pass ssDNA through a stable pore; current flowing through pore is decrease when nt passes through; there is a different signal generated for each base–>determine base seq
T cause the most current drop, followed by C, G, then A–>baseline at top
ya
what is engineered Taq?
– 5’ - 3’ exonuclease activity eliminated by point mutation or deletion
– F667Y mutation reduces preference for dNTPs over ddNTPs or fluorescent analogs
