Core genetic engineering techniques

Question 1

How many types of endonucleases are there? Which one is most commmonly used in the lab and what do that type recognise?

Answer 1

There are four types (I-IV).

II is most commonly used in the lab. The type II endonucleases recognize 4-8 bp palindromic sequences.

Question 2

What are isoshizomers?

Answer 2

Restriction enzymes that recognize and cleave at the same site.

Question 3

What are neoschizomers?

Answer 3

Restriction enzymes that recognize the same sequence but cleaves at different sites.

Question 4

Ismethylation relevant for restrication enzymes? Why / why not?

Answer 4

Yes. Because methylated DNA is generally endogenous to the organism. Non-methylated DNA is generally new DNA, maybe from a phage.

Question 5

What are DAM and DCM?

Answer 5

They are bacterial methyl-transferases. They methylate DNA, chose restriction enzymes which cleaves methylated DNA effectively if your bacterial strain is DAM/DCM positive.

Question 6

What does the klenow fragment derive form?
What’s he polymerase direction and exonuecleic direction of the klenow fragment?

Answer 6

The klenow fragment derives from DNA polymerase 1, the 5’ –> 3’ exonucleic acitivty has been removed.

Polymerase direction: 5’ –> 3’
Exonucleic direction: 3’ –> 5’

Question 7

How does T4 DNA ligase work? How do you make it avoid self-ligation?

Answer 7

With either blunt ends or overhangs; 3’ hydroxy- and 5’ phosphate groups get bound by a phosphodiester bond. The reaction is slower if there are blunt ends.

Self-ligation is avoided by adding a phosphatase, which removes 5’ phosphate (directly after restriction enzyme cleavage).

The resulting nick in the plasmid will be repaired when the plasmid is introduced to bacteria.

Question 8

Explain the work flow of incorporating an insert into a plasmid using Type IIs (golden gate) assembly

Answer 8

1. 2 inserts are designed with (1) restriction sites A and B, and (2) restriction sites B and C. Vector is ordered with restriction enzyme A and C cleavage sites.
2. Inserts are amplified by PCR.
3. Inserts, vector, restriction enzymes and T4 DNA ligase are placed in ONE tube.
4. Restriction enzymes digest the inserts and the vector, inserts are ligated to the vector and to each other by T4 DNA ligase.

Only fully digested DNA will be circular, without ligations between vector <–>insert1 <—> insert2 <–> vector, the DNA will be linear.

Question 9

Explain the mechanism of ligation-independent cloning (LIC)

Answer 9

1. Inserts are designed with long regions which are complementary to the vector sequence which is digested in 3.
2. Restriction enzyme linearizes the vector.
3. T4 DNA pol has a 5’ –> 3’ exonuclease activity in the absence of nucleotides. A small [dNTP] is added at the position where the digestion is supposed to stop (where 12-15 nt overhang has been achieved). This digestion is performed for inserts and the vector.
4. Insert ligates to vector by it having been amplified by PCR with primers that host long complementary regions to the vector.
5. Nicks are repaired whne the vector (w/ inserts) is transformed into bacteria.

Question 10

What is SLIC? Explain the mechanism.

Answer 10

SLIC = Sequence and ligation independent cloning.

SLIC utilizes the same principle as LIC, but for multiple inserts. The inserts get digested in a similar manner to the way that the vector gets digested in LIC, then the inserts ligate due to their large overhangs.

Question 11

What’s a Gibson assembly? Explain the mechanism.

Answer 11

A gibson assembly is the hybridisation of two dsDNA strands which contain the same sequence at different ends of the molecule.

1. Exonuclease digests 5’ ends
2. Fragments anneal with large overhangs
3. Polymerase extends the annealed strands.
4. DNA ligase igates the strands.

Question 12

Explain the mechanism behind TOPO cloning.

Answer 12

Topoisomerase can function as a ligase and restriction enzyme. Topoisomerase binds to 3’ Thymine which is easily outcompeted by the formation of a phosphodiester bond.

1. Taq Pol is used to amplify a DNA sequence with A overhangs.
2. The topoisomerase (which is conjugated to the vector) identifies the Adenosine overhags on the inserts, TOPO facilitates the formation of a phosphodiester bond.
3. Topoisomerase’s interaction with the vector is outcompeted by the formation of the phosphodiester bond, sealing the vector.

Question 13

What’s the principle behind Cre-LoxP recombination?

Answer 13

Cre is a recombinase that recombines elements between two LoxP sites. Depending on the orientation of the LoxP sites, deletions, insertions, substitutions and trasnlocations can be induced.

Question 14

What traits are important for good genetic vectors?

Answer 14

1. High copy number
2. Selectable marker(s)
3. Restriction sites
4. < 10kb

Question 15

How can phages be used as vectors?

Answer 15

1. Incorporate genes in the phage viriome.
2. Let phage infect bacteria.
3. Phage viriome gets circulized by cos-genes.
4. Bacterias constituively expresses the sequence of interest as a plasmid.