L16 - Alternative gene cloning methods

Question 1

describe the basic work flow of conventional restriction enzyme-based cloning

Answer 1

1. Gene of interest is amplified by PCR
2. insertion into a plasmid with a MCS and restrction enzymes
3. transfromation of bacterial cell
4. colony selection
5. plasmid isolation
6. verification by sequencing

Question 2

what are some limitations of conventional cloning strategies

Answer 2

no unique restriction sites around the target DNA sequence

can leave ‘scars’ additional nucletides comapred to prior to cloning

= when you use RE your forced to include their recognition sequences = these sites may not match up perfectly at junction

a few non-native nucleotides may remain between the 2 genes your wishing to add together

Question 3

what are the 2 types of alternatice cloning strategies and give some examples

Answer 3

ligation dependant:
Gibson assembly
TA cloning

ligation independant;
TOPO-TA cloning

Question 4

what is TA cloning

Answer 4

alternative ligation dependant cloning method

uses taq polymerases preference to accidentally add a non-templated 3’ A nucleotide in PCR when amplifying target gene

= vector chosen is linearized and contains 3’ T end
= the T and A can anneal allowing time for ligation to occor

Question 5

what does taq polymerase have that makes it useful in TA cloning

Answer 5

terminal transferase activity

= accidentally adds single NON-TEMPLATED A at 3’ end in PCR

linearized vector designed to have 3’ T overhangs for annealing

Question 6

why is normal TA cloning not directional ?

Answer 6

just As and Ts at 3’ ends that allow the annealing

= does not matter which direction the gene is inserted

Question 7

key difference between convetional and alternative cloning strategies

Answer 7

conventional uses Restriction endonucleases

Question 8

what is TOPO_TA cloning

Answer 8

alternative ligase independent cloning method

advanced version of TA cloning with the same principles of the 3’ A and T overhangs annealing

= difference is vector comes with Topoisomerase 1 attached to the cut site
= catalyses the ligation WITHOUT ligase

still not directional

Question 9

does TA cloning leave a scar or is it seamless

Answer 9

both TA and TOPO-TA leave scars

= the 3’ A and T remain

Question 10

pros and cons of TA cloning

Answer 10

Pros:
specific binding between the T/A overhangs

very fast –> TOPA TA even faster

Cons:
leaves T/A scar

cloning is not directional

limited choice of vectors and not good for multiple frag cloning

Question 11

what are the 2 ways to undergo seamless/scarless cloning

Answer 11

both are alternative cloning methods:

1.Gibson = ligation dependant

LIC. = ligation independent

Question 12

what is and describe gibson assembly

Answer 12

alternative ligation dependant SEAMLESS cloning method

= can be single or multiple fragments

1. amplify desired fragments with PCR using primers that add 15-50 nucleotides of overlapping sequnces
2. mix insert + plasmid with Gibson assembly master mix
3. 5’ exonuclease chews back = 3’ overhangs
4. overhangs anneal and polymerase fills in gaps

= no scars at junction as no RE or T/A are used

Question 13

what does gibson assembly rely on

Answer 13

overlapping 15-40nt complimentary regions

between plasmid and the fragments

= these anneal andpolymeras fills in gap = seamless

Question 14

can gibson asembly do multiple fragments

Answer 14

yes

more complex in terms of design of primers to create overlapping regions between the fragments

Question 15

pros and cons of Gibson assembly cloning

Answer 15

Pros:
directional cloning

seamless

Cons:
very expensive and complex design of primers ESPECIALLY when using multiple fragments

efficiency decreases with number of fragments

Question 16

describe what is and how LIC (ligation independent cloning) works

Answer 16

ligase independent alternative SEAMLESS cloning strategy

1. digest the vector using T4 polymerase 3’–>5’ exonuclease activity to the first A to create 5’ overhangs
2. create ‘homology arms’ on insert via PCR to create complementarity between vector and insert
3. digest insert with T4 3’–>5’ exonuclease activity to create 5’ overhangs to the first T in sequence

4.mix vector and insert in tube
5. anneal due to complimentarity
6. insert into bacteria to use cells repair machinery to fix nick

= ligase INDEPENDANT

Question 17

how and why do we digest the vector to the first ‘A’ in sequence and insert to first ‘T’ for LIC

Answer 17

we add in a specific nucleotide (dNTP) with the T4 polymerase

= creates equilibrium between the nucleotide being added on and taken off at the first site of this nucleotide in sequence

This limits the ‘chewing back’ of the exonuclease

Question 18

simple steps of LIC - not detail

Answer 18

1. linearize vector via restriction enzyme
2. create 5’ overhangs with T4 and dTTP
3. create insert with PCR and homology arms
4. create 5’ overhangs with T4 and dATP
5. mix in tube
6. annealing of insert and vector
7. transform into bacterial cell
8. nick healed by host cell

Question 19

Pros and cons of LIC

Answer 19

Pros:
seamless

multiple fragments can be used in a single step

Cons:
may not be a unique dNTP in sequence = not able to use T4 pol to make compatible ends

limited sequence modifications available = only adding the single dNTP at 3’ end of vector = cannot add codons or something to affect expression