L4 Flashcards
how large is the genome size of E.coli
4.7 Mb
how many non coding regions does E.coli have
4 288
how large is the human genome
3.2 Gb
how many non coding regions do have
21 000
what is saccharomyces cerevisiae
yeast
what is caenorhabditis elegans
worm
what is drosophilia melanogaster
fly
what is arabidopsis thaliana
weed
What is Maxam Gilbert sequencing
base specific chemical cleavage; first method developed used in 1970s and 1980s, Sanger method became the preferred option
Describe the Maxam Gilbert sequencing
- ) supply a single pure template of DNA to sequence
- ) nucleotide labeling system for sensitive DNA detection (radioactivity and fluorescent labeling etc)
- ) Supply a sequencing primer, ~ 20 bp ss DNA
true or false, the sequence of the primer must be known (“Catch 22”) - you can only sequence DNA if you already know some sequence
true
How do you solve the problem in which the primer must be known before sequencing of DNA can occur
- ligate your unknown fragment to a known sequence eg. clone into plasmid or ligate oligonucleoties
1. ) “Universal” sequencing primers were designed that anneal adjacent to the multiple cloning sites of common cloning vectors - primer ‘reads’ 500-1000 bp of sequence of any insert cloned in MCS
2. ) Ligate known sequence to each end - randomly fragment target DNA for sequencing
- ligate oligonucleotides of known sequence to ends
Describe the Sanger method
Dideoxy chain termination
- incorporation of ddNTP lead to termination of nascent chain because lacks 3’OH; cannot form a phosphodiester bond with incoming dNTP
true or false, automated sequencing, fluorescent labelling of ddNTPs gives 500-1000 bp of sequence per reaction
true ; use taq polymerase
what are the advantages of fluorescent labeling and laser reading compared to original radioactive labeling and radioautography
- simaltaneously label all 4 bases
- safer as non radioactive
- computer automated calling: faster scoring, fewer errors
true or false, initial sequence close to the primer is always tough to read
true
true or false, sequence further out from the primer gets harder to read due to diffusion of bands, less consistent incorporation of dideoxynucleoties
true
what is capillary electrophoresis
- this allowed further automation as automated sampling loading
- occurs in a small scale, faster than electrophoresis, higher throughput
AB13700: 6 runs/day, 96 samples at a time, 700 bp per run, 500 000 bp per machine per day
what is pyrosequencing
- developed in the 1990s
- sequential addition of bases one at a time to template+primer+polymerase
- generates flash of light enzymatically when a base is added
- cheap, fast, automatable
- about 4-500 bp per run
in pyrosequencing what causes the flash of light
CTP coding enzyme which means the original base was a G
Describe the pyrosequencer Roche 454; generally flood with the base and see where the flash of light will come from
- new high throughput machine released in 2005
- based on pyrosequencing
- main advantage is higher throughput because of massively parallel reactions ( 1M vs 96 with Sanger) via emulsion PCR
- DNA fragmented , linkers of known sequence added, single molecules attached to beads, PCR on individual beads in aqueous droplets in oil emulsion, sequencing in micropore, sytem ( bead per well)
- efficient, cheap, reads slightly shorter (500 bp), homopolymer errors in reading
describe Roche 454
- ) single DNA strands are immobilized on individual beads
- ) these molecules are amplified by PCR
- ) each bead is deposited into a tiny well
- you can get a million independent fragments in one PCR - ) flood with base and see where the flash of light will come from
true or false, Roche 454 have higher error rate in estimating length of homopolymer
true
What is ion torrent
machine cost 120 000 NZD run cost $800 run time 2 hrs read length up to 200 bp reads per run 500 000 bp detection: standard bases added individually with flushing between each, detection by pH change/current (proton) using semiconductor chip
- no label- standard NTPs
- faster read times
- homopolymer error rate applies
