Sequenced Based-Cloning

Question 1

What is recombinant DNA?

Answer 1

A piece of DNA formed by joining together two or more smaller DNA molecules from different sources
= isolate, amplify and modify specific DNA sequences

AKA molecular cloning and genetic engineering

Question 2

What does recombinant DNA allow?

Answer 2

This allows us to engineer DNA molecules for our own purposes:
Therapeutic antibodies and insulin
Genome sequencing

Question 3

What is in a traditional cloning toolkit?

Answer 3

Restriction endonucleases
DNA ligase
DNA polymerases
Alkaline Phosphatases

Question 4

Describe restriction endonucleases?

Answer 4

They recognise palindromic sequences as they have 2-fold rotational symmetry (they are homodimers)
They cleave the DNA by breaking phosphodiester bonds to generate overhangs - that base pairs with complementary 5’ or 3’ overhangs called “sticky ends”
3’OH end is where primers extend from

More than 600 available commercially - to manipulated DNA
5 types - differ in the molecular structure, where they cut and if the sites are separate from each other

Most common = class II
They use Mg2+ as a cofactor

Question 5

What is a classification of some restriction endonucleases?

Answer 5

Neoschizomers - recognise the same recognition sites but cleave in slightly different sites
Isoschizomers - recognise the same recognition sites and cleave in the same sites

Question 6

What are restriction endonucleases used for in bacteria?

Answer 6

An antiviral defence system in bacteria

For every RE there is a DNA methyltransferase
They methylate the DNA bases, protecting against digestion of the RE
Therefore non-methylate DNA = foreign = degrade

Question 7

Describe DNA ligase?

Answer 7

Joins compatible DNA strands via 5’ monophosphate and 3’ hydroxyl, forming phosphodiester bonds
ATP dependent
Follows base pairing
These ends can be blunt or sticky
H-bonds must happen along the entire length for ligation to occur
They tend to originate from T4 bacteriophage

Question 8

Describe DNA polymerases?

Answer 8

Produces blunt ends by filling in 5´ overhangs or removing 3’ overhangs in the presence of nucleotides

Klenow fragment of DNA Pol I of E.coli - a pyrolytic product
It retains the polymerisation fidelity of the native enzyme without degrading the 5’ terminal
This enzyme used to be used in PCR before Taq polymerase

Question 9

Describe the alkaline phosphatases?

Answer 9

Prevents ligation (phosphodiester bonds) of the same strand
It removes the 5’ monophosphate groups from DNA
Reduces background empty ligating vectors

Question 10

Give an overview of cloning?

Answer 10

The process through which recombinant DNA molecules are constructed and amplified
An insert is placed into a vector and replicated in a host cell to produce a recombinant DNA molecule

Vectors: Plasmids, bacteriophage lamda, cosmids and bacterial artificial chromosomes

Question 11

Describe plasmid vectors?

Answer 11

Natural:
Circular double stranded DNA
<1 kb to >100 kb in size
From 1 to 1000 copies per cell
Replicate independently of chromosome

Synthetic:
Most are 3 kb pairs
Origin of replication: recruits host cell DNA replication machinary and determines compatibility with other plasmids
Selectable marker: Antibiotic resistance to the host cell
Multiple cloning site (MCS): Contains a selection of unique restriction sites that we can use to insert DNA fragments (or “inserts”)

Question 12

Describe cloning with a plasmid?

Answer 12

1. The cloning vector (in the MCS) and the foreign DNA are cut by the same restriction endonucleases
   Dephosphorylate with a phosphatase to prevent vector re-ligation
   Ligate the sticky ends of the vector with fragment with DNA ligase
   = A chimeric DNA or recombinant plasmid
2. The recombinant plasmid is added into a host bacteria
   Either chemical tranformation (e.g. treat with CaCl2 to make competent + heat shock or electroporation
3. Selection of “transformants”
   After plasmid replication and bacterial cell division
   We spread the cells on a agar plate containing ampicillin (use antibiotic resistance genes)
   Cells that took up DNA are called competent and transformers = they can grow (form colonies of cells - clones)

Question 13

Describe the pUC19 plasmid or a bluescript plasmid?

Answer 13

A Phagemid (a plasmid with phage origin)
It contains the LacZ gene - this encodes beta galactosidase, which cleaves X-gal to produce blue colonies

So if the vector contains an insert the LacZ gene is disrupted
Therefore colonies containing a genomic insert = white
Without insert = blue

Question 14

What do we have to do to the host bacteria?

Answer 14

1. Disablement (mutation) of the host for safety reasons
   Use an auxotroph: host cannot synthesise a metabolite/amino acid;
   Host can only grow on growth medium supplied in the laboratory - so they can’t escape the lab
2. We need stable maintenance of the transformed DNA
   Avoid rearrangements by using mutants in recombination genes such as recA
3. We need efficient “transformation” by plasmid DNA
   Use a host that is deficient in natural restriction modification systems

Question 15

What is PCR?

Answer 15

Polymerase chain reaction:
PCR selectively amplifies a stretch of DNA from a larger piece of DNA
This takes place in a thermocycler
Needing: template DNA, forward/reverse primers, dNTPs, Taq DNA polymerase and buffer

Denaturation - melts DNA duplexes into individual strands - ssDNA
Annealing - allows primers to anneal with template and polymerase to bind
Extension - extension of primers along the template by polymerase

Temperature - 95, 55, 72

Question 16

What should be considered with primers in PCR?

Answer 16

Too short = annealing with other regions of the template
Too long = hybridise at a slower rate and decrease efficiency of the PCR

5’ ends of primers form the outside boundary of an amplicon - don’t have to be perfectly complementary
A primer can have sequences at it’s 5’ end that aren’t involved in the first round of PCR
They will be involved in following rounds - incorporated into the amplicon from the second round onwards

3’ region of the primer must be antiparallel complementary to initiate DNA synthesis

Question 17

Describe the golden gate assembly

Answer 17

Allows a scar-less assembly of a vector and insert into a bacterial host
Type II restriction endonucleases cut outside the recognition sequences (non-palindromic)
BsaI - creates a 4 nucleotide unique overhang (any nucleotide combination) = 4^4 = 256 combinations
One restiction enzyme can be used for both ends of the fragment
Both fragments will have the same restriction site but in opposite directions, with complementary overhangs

The fragments used in the assembly are designed such that the digested fragments and the final assembly no longer contain Type IIS restriction enzyme recognition sites
Therefore no further cutting by Bsa I is possible
The annealing of complementary overhangs directs the order and orientation of fragment assembly
They are put together using T4 DNA ligase

Question 18

What are some essential features of all vectors?

Answer 18

Ability to readily incorporate “foreign” DNA
Ability to be readily introduced into host cell and selected
Ability to replicate in host cell

Question 19

Describe the bacteriophage lambda?

Answer 19

It infects E.coli
It has 2 types of cycles: lytic and lysogenic

Lytic - bacteriophage uses the cells machinary to produce nucleic acids/proteins (packaged to form viral particles), before the cell bursts open to release thr virons - killing the cell

Lysogenic - bacteriophage incorporates the viral nucleic acid into the host genome
Viral DNA is replicated along with the host DNA

Question 20

Describe a bacteriophage infecting E.coli?

Answer 20

The phage attaches to the E.coli cell membrane using receptors
DNA is ejected through the tail, that was store in the head, into the E.coli (conduction) - this DNA could be recombinant that we edited
DNA present in phage has a single-stranded overhang of 12 nt, which are complementary to each other
Once in the host bacteria the DNA can base pair and be closed by DNA ligase into a circular piece of DNA

If there are favourable conditons it will undergo the lysogenic cycle - copying both DNAs and then extract the DNA we want to amplify by breaking down the cell
Until the conditions revert it to the lytic state

More likely it will adopt the lytic cycle
DNA replication and synthesis of phage products/particle assemble and then cell lysis - releasing the phages

Question 21

Describe further the lytic cycle of a bacteriophage?

Answer 21

The DNA is replicated in a unidirectional ‘rolling circle’ mechanism
Producing linear genome cos sites

Terminase - results in linear DNA between the cos sites being packaged into a pre-head structure of the capsid
Filled heads then associate with pre formed tails = infectious particles

Question 22

What are some assays of bacteriophages?

Answer 22

Liquid culture assay: Add phage to bacterial culture
If enough bacteria are lysed it change colour (clearer)
If incubated long enough we may see bacteria that has been integrated through lysogeny (colour returns)

Plaque assay: Add phage to bacterial culture and spread on plaques
If the bacteria are in excess - a lawn of bacterial growth will be present on the agar plate
At positions where cells infected were inoculated - a plague of area of dead cells will appear
If during infection in the plague a lysogen is produced the plague could look clearer

Question 23

What are some constrainst of using lambda as a cloning vector?

Answer 23

Only DNA molecules whose length is 78-105% that of wild-type lambda, which is 48.5 kb, may be packaged into phage head
The constraints limit size of a recombinant construct to 37 to 51 kb
Therefore can only add 2.5 kb without removing part of original genome – not advantageous compared to plasmids
However, part of lambda can be removed, i.e. genes required for lysogenic cycle, increasing the size of DNA that can be cloned

Question 24

What are the types of bacteriophage lambda vectors?

Answer 24

Replacement vectors

Insertion vectors

Question 25

Describe a bacteriophage lambda, insertion vector?

Answer 25

No lower limit on size of insert when using insertion vector
The empty vector can be propagated in E. Coli
The central region is only required for the lysogenic cycle
Usually suitable for cloning of fragments of 0 - 10 kb, depending on vector
Cleavage at X produces two arms are then ligated with an insert to form a recombinant.
It has the complementary overhangs - that will anneal to form a recombinant concatemer

Question 26

What is a concatemer?

Answer 26

Concatemer - a long continuous DNA molecule that contains multiple copies of the same DNA sequence linked in series

Too long to undergo transformation into a bacterial host cell
Not a pitfall - can be packaged in vitro
Kits include empty pre-heads, talks and packaging proteins

Question 27

Describe bacteriophage lambda replacement vectors?

Answer 27

To create this vector - a larger portion of the central non-essential region of the lambda is substituted with a ‘stuffer’ fragment (up to 20 kb)
The stuffer allows propagation into E.coli
During cloning the central ‘stuffer’ fragment is first removed and then replaced by a larger foreign insert (up to 22 kb)
Stuffer DNA = replaceable DNA, that doesn’t derive from the lambda
It has been engineered to contain additional restriction sites to prevent reinsertion/annealing during cloning

This isn’t useful for cloning small molecules

Question 28

Describe the vector - cosmids?

Answer 28

They are plasmids that contain the lambda Cos site (used in lambda in vitro packaging)
In vitro packaging extracts will package any molecule that carries cos sites separated by 37 – 50 kb

Cosmid libraries - we propagate cosmid vector as plasmid and cleave with a restriction enzyme, add foreign DNA and ligate to form concatemers (not circular products)
Recombinant cosmid lacks genes required for lambda particle production
Therefore colonies are produced on ampicillin plates

Question 29

What are some limitations of traditional cloning?

Answer 29

“Cutting and pasting” steps are separate reactions - usually restricted to the joining of only two DNA fragments, vector plus insert, in a single ligation
Additional rounds of the complete cloning cycle are normally used to join an additional fragment, e.g. a second insert
Construction of recombinant molecules can be limited by the availability of suitable restriction enzyme sites and the presence of restriction enzyme sites in the middle of fragments
The joining of large fragments can be problematic due to the lower concentration of ends

Question 30

What is Gibson Assembly?

Answer 30

A cloning technique that doesn’t use restiction enzymes

Also called isothermal assembly

Question 31

What are the benefits of Gibson Assembly?

Answer 31

Can assemble multiple fragments in a single reaction
Some of the fragments can be long, >3 kb
The cloning can be “scarless”, i.e. restriction enzyme recognition sites are not left at the junctions of jointed fragments
Any insert fragment with appropriate overhangs can be efficiently ligated into any plasmid backbone including those designed for use in other cloning approaches
Is not limited by the same factors that limit traditional cloning

Question 32

What is the mechanism of Gibson Assembly?

Answer 32

The ends need to share a short sequence (15-20 bp) and when the fragments are joined only one copy of this is retained

1. A 5ʹ-exonuclease activity produces single-stranded segments called 3′ overhangs
2. 3′ overhangs that are complementary base-pair, in effect producing pairs of primer and template
3. In a self-priming reaction, the DNA polymerase activity then extends from the 3′ end of the overhangs now annealed to a complementary DNA to fill in the gaps
4. A DNA ligase then seals the nick and covalently links the DNA fragments together

The fragments join in only one order and one direction