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
ABI DNA seq?
denature, anneal, extend; dye-labelled terminators fluorescence diff colours–>run on gel; OR can have the first nt of a segment labelled different colours based on the ddNTP present in the mix; generate wavelength chart of comp, get diff colour each wavelegth pattern, –>that’s the nt
describe pyroseq/454
DNAfragments(ss)aremixedwithagarosebeads carrying oligonucleotides complementary to the adaptors under conditions to have one fragment per bead
• Clonalamplificationby“emulsionPCR”
• Each bead is placed into one tiny well in pico titer plate (PTP), and the DNA it carries is used as template for sequencing
• Oninstrument,thePTPactsasaflowcellintowhich each pure nucleotide solution is added step-by-step
to seq, add nt to oligo on bead via polymerase; PPi will be generated; add PPi to APS–>get ATP and sulfate via sulfurylase; with the new ATP, combined with luciferin and O2–>get AMP + PPi+oxyluciferin and LIGHT(via luciferase) (supply one dNTP at a time)
–look at results on a pyrogram (shown on slide 36 of wang 03 pt)
for sanger seq–put into vectors then use primers which bind to the regions flanking the MCS–>can’t bind primers to actual DNA seq, as you are trying to determine it! called universal primers
ya
• Taq DNA polymerase
DNA Polymerases
– Thermostable: sequencing reactions at elevated temperatures reduces problems with templates rich in secondary structures
– 5’ -3’ exonuclease activity
– Preference for dNTPs over ddNTPs or fluorescent analogs (factor of ~ 103)
ya
second gen seqs?
pyro, illumina/solexa, ion torrent
describe illumina seq
prepare libraries of short DNA frags–>link to adapters; attach the ssDNA to a solid phase via adapter seq; clonal amplification via bridge PCR; sequence by synthesis using the DNA bound to solid phase–>clustered on solid phase; to sequence, one type of fluorescently-labelled nt is added at a time and the 3’OH is blocked (reversible terminator)–>excite DNA and cleave off fluorophore; get light–>if the DNA pieces cluster together, you will get the same colour but a lot of light in that one little spot
second gen seq disadvantages?
SGS technologies: High throughput and lower cost but:
• Template preparation – mostly PCR based - bias in template representation
- procedures can be technically difficult
• Shorter read (most of them, but improving)
- signal depends on average of many molecules - “dephasing” decreases read length
• More errors
• Use “wash and scan” approach - pausing at each step
RES–Classified based on subunit composition, co-factor requirements and DNA-cleavage properties
Type II
- usually homodimer, recognize/cleave DNA at the same site
Type IIS
- Asymmetric recognition site with cutting at a defined distance e.g. FokI
Type III
- Asymetric recognition site; cuts approximately 26 bases away from the recognition sequence e.g. EcoP15I
ya
complete genomics adv and disadv?
Advantages
• High throughput; low reagent cost • High accuracy
Disadvantages
• Very short read ~ 10 bases
• Template preparation: labor intensive and difficult
