L3 (2) synthesizing DNA

Question 1

chemical synthesis

Answer 1

• put multiple DNA molecules on surface
  -> add one base on each at a time
• 99,5% efficient => 1/200 is wrong
  = bad percentage!

Question 2

oligonucleotide synthesis

Answer 2

• happens 3’ -> 5’
• one bead => 200 micrograms oligonucleotide synthesised
  -> molecule averge 30 nts
  -> synthesize 10 000 spots with 1 ng each

Question 3

oligonucleotide synthesis error rate calculate

Answer 3

(percentage)^n
- n= amount of nts

Question 4

what if primer contains error?

Answer 4

20/20 correct => yay!
19/20 => would probably still transcribe
- less than that is very meh
- the closer to the end that the error is the more likely transcription is to stop

Question 5

how to synthesize entire (short) gene

Answer 5

• 2 primers with 3’-ends complementary to each other
• DNAP will replicate on both primers to create dsDNA

Question 6

how to create longer fragments of dsDNA

Answer 6

= oligo overlap method = overlap extension assembly
- have up to 8 oligos with DNAP replicating on each of them
- when DNAP reaches next oligo => falls off
- the “nicks” created in between oligos are sealed with DNA ligase

Question 7

possible errors of oligonucleotide synthesis

Answer 7

• one nt missing = most common
• one nt modified in some way
• one nt chemically changed to another one

Question 8

how to purify DNA fragments

Answer 8

• transformation into E. coli
• using MutS
• MutS will recognize error => MutL gets there and cut off error
• DNA with mutations can be physically filtered because they have MutS bound to them
• reduces errors to 1/10000

Question 9

assembly of DNA, old way

Answer 9

• consecutive assembly
• restriction enzymes cut open plasmid => gene can be added

Question 10

restriction enzymes

Answer 10

• recognize 4,6,8 nts so if DNA long enough the sequence can show up often => cuts wrong spot
• defends against phages by binding to both sides of dsDNA
• host’s DNA methylated so they only bind to virus’ DNA

Question 11

Gibson assembly

Answer 11

• simultaneous
• have 2 dsDNA overlapping
  -> T5 exonuclease cuts 5’-end
  -> 3’-ends overlap
  -> at 50 degrees Celcius they anneal with the help of Phusion polymerase and Taq ligase, T5 exonuclease is gone
  => one long fragment
• no restriction enzymes
• limited to 8 oligos because of risk of error