L4

Question 1

how large is the genome size of E.coli

Answer 1

4.7 Mb

Question 2

how many non coding regions does E.coli have

Answer 2

4 288

Question 3

how large is the human genome

Answer 3

3.2 Gb

Question 4

how many non coding regions do have

Answer 4

21 000

Question 5

what is saccharomyces cerevisiae

Answer 5

yeast

Question 6

what is caenorhabditis elegans

Answer 6

worm

Question 7

what is drosophilia melanogaster

Answer 7

fly

Question 8

what is arabidopsis thaliana

Answer 8

weed

Question 9

What is Maxam Gilbert sequencing

Answer 9

base specific chemical cleavage; first method developed used in 1970s and 1980s, Sanger method became the preferred option

Question 10

Describe the Maxam Gilbert sequencing

Answer 10

1. ) supply a single pure template of DNA to sequence
2. ) nucleotide labeling system for sensitive DNA detection (radioactivity and fluorescent labeling etc)
3. ) Supply a sequencing primer, ~ 20 bp ss DNA

Question 11

true or false, the sequence of the primer must be known (“Catch 22”) - you can only sequence DNA if you already know some sequence

Answer 11

true

Question 12

How do you solve the problem in which the primer must be known before sequencing of DNA can occur

Answer 12

• 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

Question 13

Describe the Sanger method

Answer 13

Dideoxy chain termination
- incorporation of ddNTP lead to termination of nascent chain because lacks 3’OH; cannot form a phosphodiester bond with incoming dNTP

Question 14

true or false, automated sequencing, fluorescent labelling of ddNTPs gives 500-1000 bp of sequence per reaction

Answer 14

true ; use taq polymerase

Question 15

what are the advantages of fluorescent labeling and laser reading compared to original radioactive labeling and radioautography

Answer 15

• simaltaneously label all 4 bases
• safer as non radioactive
• computer automated calling: faster scoring, fewer errors

Question 16

true or false, initial sequence close to the primer is always tough to read

Answer 16

true

Question 17

true or false, sequence further out from the primer gets harder to read due to diffusion of bands, less consistent incorporation of dideoxynucleoties

Answer 17

true

Question 18

what is capillary electrophoresis

Answer 18

• 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

Question 19

what is pyrosequencing

Answer 19

• 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

Question 20

in pyrosequencing what causes the flash of light

Answer 20

CTP coding enzyme which means the original base was a G

Question 21

Describe the pyrosequencer Roche 454; generally flood with the base and see where the flash of light will come from

Answer 21

• 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

Question 22

describe Roche 454

Answer 22

1. ) single DNA strands are immobilized on individual beads
2. ) these molecules are amplified by PCR
3. ) each bead is deposited into a tiny well
   - you can get a million independent fragments in one PCR
4. ) flood with base and see where the flash of light will come from

Question 23

true or false, Roche 454 have higher error rate in estimating length of homopolymer

Answer 23

true

Question 24

What is ion torrent

Answer 24

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

Question 25

What is nanopore sequencing

Answer 25

• single molecules read out
• measures changes in electrical signals as DNA snakes through the tiny protein holes or pores on a silicon chip (ie) single molecule sequencing, very fast, low cost
• claimed to detect methylated C bases directly

Question 26

length of Sanger

Answer 26

1000

Question 27

number of runs of sanger

Answer 27

10^3

Question 28

Roche 454 (pyro) length

Answer 28

7-800 bp

Question 29

Roche 454 number of runs

Answer 29

10^7

Question 30

Ion torrent length

Answer 30

300 bp

Question 31

number of runs of ion torrent

Answer 31

10^7

Question 32

nanopore length

Answer 32

40 000

Question 33

number of runs of nanopore

Answer 33

10^6

Question 34

true or false, computers identify regions of identical sequences overlap and assemble these into “contigs”, sequence is more accurate if it is obtained in both directions

Answer 34

true

Question 35

what is a contig

Answer 35

computer assembled sequence

Question 36

what is the expected frequency of a stop codon in random sequence

Answer 36

1/64 bases

Question 37

how are genes identified in bacteria

Answer 37

by finding open reading frames