Week 5 manipulation of DNA, Ligases

Question 1

What do ligases do?

Answer 1

They join adjacent nucleotides by forming phosphodiester bonds, they do not synthesize new DNA

Question 2

List some general info about Ligases

Answer 2

Ligases aren’t site specific
Ligation of sticky ends is more efficient than ligation of blunt ends.
T4 phage DNA ligase is most common ligase uses ATP as co-factor

Question 3

RE’s are used to cut into DNA but what does it leave behind to allow the strain the be rejoined

Answer 3

It leaves 5’P and 3’OH ends that can be ligated together

Question 4

List some pros and cons with a Blunt ended DNA molecule

Answer 4

Cannot direct attach to a sticky ended molecule, so a sticky end need to be attached with polymerase or taken of the sticky with nuclease.

Blunt ended molecules don’t need to be cut with the same enzyme however a Restriction site will only be created if the same enzyme is used on both molecules.

Question 5

What are linkers used for?

Answer 5

Short ds DNA containing a restriction site, are added to blunt ends. Restriction digestion leave a single linker on each end, is limited to DNA that do not contain a recognition site for the enzyme used

Question 6

What are Adaptors?

Answer 6

Are short synthetic oligonucleotides that are designed to already have a sticky end, these are attached to blunt ended molecules.

They have the 5’-P removed to prevent the ligation of adaptor to adaptor.

After adaptors are ligated to blunt ended molecule the 5’-P is returned so cloning can occur this is done with the enzyme polynucleotide kinase.

Question 7

List some alternatives to using RE’s and T4 DNA ligase

Answer 7

TOPO - cloning
Gateway cloning
Recombineering
Gibson assembly

Question 8

TOPO cloning

Answer 8

Useful for cloning DNA produced by PCR
Oligo used in PCR are non-phosphorlated just like the 5’ ends on amplifed DNA

Rapid - high efficiency
Cons - vector from commercial supplier

Question 9

Recombineering

Answer 9

Cloning without RE, needs specific strains or cloning hosts, can be done in vitro

Pros - Rapid, good at assembling complex or large DNA fragments
Cons - not cheap or easy

Question 10

Gibson assembly

Answer 10

used for cloning a single or multiple fragments into a vector, in vitro technique, good for cloning operon or longer sections of DNA