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AMB > LECTURE 4 (Cloning) > Flashcards

LECTURE 4 (Cloning) Flashcards

1
Q

What is a clone?

A

A genetically identical copy

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2
Q

What are the two main steps of cloning?

A
    1. generate inserts and ends

- 2. ligation of the inserts via phosphodiester bonds

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3
Q

What is an insert?

A

A DNA fragments that’s gonna be cloned

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4
Q

What is single cloning?

A

cut of the plasmid with a RE, open another plasmid and insert the insert you cut off from the first plasmid. The insert is joined into the new plasmid using a ligase.

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5
Q

Problems of single cloning? (2)

A
  1. the vector: the mineralization of a vector could end up in empty vectors since they’re highly compatible
  2. The insert obtained can go in either direction and some genes might not be expressed
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6
Q

How do we solve the religation problem? What happens with the compound that is added?

A

ligation depends on a p in the 5’ end. alkaline phosphatase can remove this p group to avoid religation. only one p can end up in ligation although there would be a DNA nick that would be repaired later on.

Alkaline phosphatase must be removed before ligation with phenol extraction (to remove proteins) and ethanol precipitation (remove contaminants)

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7
Q

What is double cloning?

A

efficient methods w/o the problems encountered in single cloning. The vector is digested with 2 different restriction enzymes therefore no ligation of the vector bc the ends are incompatible and no mis-oriented insert

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8
Q

Application of BamHI and BglII in cloning.

A

you can have an insert cub by BamHI and a vector cut by BglII and they’d be compatible.

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9
Q

Production of blunt ends in cloning, what for?

A

they’re universally compatible however there’s the orientation problem plus ligating blunt ends is hard.

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10
Q

What is the transformation principle?

A

The insertion of the clone vector into an organism, it’s not very efficient but antibiotics can be added to a plasmid so only the resistant ones in a medium would grow.

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11
Q

Problems with double cloning (2)?

A
  • the conditions should be suitable for both RE

- maybe the sites are very close together then the cut would be inefficient

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12
Q

How can we solve the orientation cloning?

A

either by carrying double cloning or by using a site that is asymmetrically placed within the insert to check its orientation

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13
Q

Cloning sites in plasmids? What are MCS?

A

Where RE cut, must not be located close to the ori. MCS are sites that recognizes several enzymes

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14
Q

How can we modificate fragment ends?

A
  • covert them into blunt ends by filling the complementary strand somehow
  • use linkers and adapters
  • use terminal transferase with dNTP that will repetitively add that specific dNTP to the strand adv: no relegation diasv: a high [dNTP] is needed.
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15
Q

What are linkers? What are adapters?

A

linkers: short pieces of DNA that contain a restriction site, we can join them to a blunt product to get s RS there
adapters: they’re used if the insert already has a RS. the adapters are short pieces of DNA that will anneal together to create a dsDNA fragment w/ different sticky ends or one sticky and one blunt.

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16
Q

What is the golden gate assembly?

A

we use an RE that recognizes a specific sequence but it cuts next door. You can create compatible ends of different inserts and then ligate them all into the final vector

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17
Q

Why is BsaI used in GG assembly?

A

It’s used to make them artificially and put them at one end of the primer.

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18
Q

say 2 adv and 2 disadv of GG assembly?

A

adv: infinite modularity
easy to form libraries

diadv: BsaI is needed RS seq must not be palindromic and the overhang seq must differ

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19
Q

What is T/A cloning?

A

exploits Taq polymerase which adds an adenosine at the end of the insert, by creating vectors with T as overhangs this would ligate. can be done with PCR

20
Q

What is T-cloning vector?

A

similar to T/A cloning, explots topoisomerase. You create a vector with topoisomerase bound (via protein-phosphate link) then you add an insert with A and ligase it’s not even needed. vector won’t ligate because ends are not compatible.

21
Q

via which mechanism is topoisomerase bound to a vector?

A

Protein-phosphate link.

22
Q

What are topoisomerases for?

A

check the degree of supercoiling and can act as ligases joining two DNA molecules

23
Q

Topoisomerase I and vaccinia virus.

A

this is used for cloning bc topo I recognizes the seq TTCCC therefore there’s a T overhang and a vector with A overhang can be made to join them.

24
Q

What does Soeing means?

A

Synthesis overlap extension, it’s a PCR based cloning method. It clones a insert of choice into a vector of choice.

25
Q

What doesn’t Soeing use?

A

It doesn’t use ligases or RE.

26
Q

How does soeing work?

A

We have the plasmid and the insert (A and B) in PCR. We add chimeric primers that will extend both A and B to form a homology region respectively. Tª increases and both fragments will denature. Tª decreases and then we find annealing. The insert is extended by fusion DNAP until it reaches the 5’ end of the vector. in the end we find a plasmid with two nicks. DpnI will digest the parental plasmid.

27
Q

When does T4 DNAP have a exonuclease activity?

A

when there’re not dNTPs to add, adding any dNTP will stop the exonuclease activity.

28
Q

Describe SLIC

A

You have an insert and a vector with homology seq. you add T4DNAP that will chop off the ends of the 3’ to 5’ strand, then you add dNTPs and vector will anneal with the insert to get the recombinant plasmid.

29
Q

What is the difference between SLIC and Gibson?

A

Gibson uses a specific exonuclease and ligases to seal the nicks rather than T4 DNAP. It also work better for longer sequences

30
Q

What is de novo artificial synthesis?

A

It’s a way of creating artificial genes through a computer

31
Q

What is Cre-lox recombination?

A

It’s another type of cloning in which the Cre recombinase acts specifically in the lox site (locus of crossing over of bacteriophage P1)

32
Q

In which loning strategies the products of ligation may have inserts in either “forward” or “reverse” orientation? (3)

A
  • TA cloning
  • single
  • blunt ended.
33
Q

In what way topo cloning differs? (2)

A
  • no ligase is used

- Topo is used to join the two DNA substrates of the ‘ligation’ reaction

34
Q

Who invented and when gateway cloning?

A

Invitrogen invented it in the 90s

35
Q

Why is PCR used for cloning?

A

If we have a whole sequence we should take primers and amplify the region of interest

36
Q

4 Characteristic of gateway cloning.

A
  • faster
  • 90% effective
  • universal (all types of DNA fragments)
  • available for several organisms
37
Q

What are the 3 things needed for cloning?

A

it uses recombination (recombinases) and att recomb sites + uses clonases

38
Q

What is gateway for?

A
  • analysis of genes and proteins

- cloning and subcloning.

39
Q

Steps of gateway cloning.

A
  • We need to put our gene of interest into a vector to create the empty clone (this is done through gateway)
  • att seq of entry clone and of destination vector match and are recombined by LR clonase
  • you have L in entry clone and R in destination vector (needs LR clonase)
  • you feet in the end B in the expression clone and P in the by-product
  • you can carry the reaction in reverse by using BP clonase in order to shuffle things backwards or put the final clone into another vector
40
Q

How can a entry clone be created? (2)

A
  • using RE and ligases

- in vitro recombinase of PCR products

41
Q

What is an entry clone?

A

clone carrying our gene of interest and should have some sort of antibiotic resistance. IT HAS ATT (RECOMBINATION SITES) ON BOTH SIDES OF THE GENE. It can be cloned in different destination vectors

42
Q

Why are att important? What are att?

A

They’re recombination sites that derive from viruses. They’re on both sides of gene of interest and have and sticky end that will match with the sticky end of the gene of interest.

43
Q

What is the main problem with gateway cloning?

A

Recombinase is very expensive.

44
Q

What is the by product in gateway?

A

The rest that hasn’t been used

45
Q

What is ccdb for?

A

It’s a gene in the destination vector that stops the by-product from growing

AMB (12 decks)

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