Recombinant DNA techniques

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 methods allow us to acquire DNA segments?

Answer 3

Reverse transcriptase, RNA into cDNA

Restriction endonucleases:
cleave phosphodiester bonds at internal sites, generating smaller DNA fragments

Question 4

Describe restriction endonucleases?

Answer 4

They recognise palindromic sequences as they have 2-fold rotational symmetry (they are homodimers)
The RENs cleaves the DNA to generate overhangs - that base pairs with complementary overhangs from other fragments called “sticky ends”

Most common = class II
They use Mg2+ as a cofactor
They are very specific to cleave the correct DNA sequence

Question 5

What are restriction endonucleases used for in bacteria?

Answer 5

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 6

How can we isolate the DNA sequences?

Answer 6

Gel electrophoresis
DNA travels through a gel mesh, moved by a repulsion to voltage
They start at the negative end and move to the positive electrode
The further down the gel = smaller size

Agarose = larger pore size (dsDNA 0.1 to 60kbp)
Polyacrylamide = smaller pore size (5-1000nt)

Question 7

What is cloning?

Answer 7

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

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

Question 8

Describe plasmids?

Answer 8

Most common vector
Circular DNA that can replicate autonomously within the host cell

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 9

Give an overview of cloning with a plasmid?

Answer 9

The cloning vector and the foreign DNA are cut by the same restriction endonuclease
DNA is amplified by PCR if needed
The sticky ends of the vector and the foreign DNA fragments anneal and are covalently joined by DNA ligase
= A chimeric DNA called a recombinant plasmid
The recombinant plasmid is added into a bacteria - transformation

Question 10

Describe bacteriophages?

Answer 10

Phages are viruses that infect bacteria
AKA lambda phage
An alternative cloning vector

A bacteriophage is commonly used as a cloning vector as it has 2 types of life cycle: lytic and lysogenic
We can remove lysogenic genes and replace them with something else
Viral DNA packed into proheads which induces head maturation

Question 11

Give an overview of cloning with a bacteriophage?

Answer 11

1. Virus infects E coli cell and replicates inside it
2. The cell lyses, releasing millions of identical progeny virus particles
3. Progeny virions infect the surrounding E coli cells in the lawn
4. This creates a hole or plaque in the lawn

Each plague corresponds to a different viral clone (i.e. insert from the library)
This allows us to simultaneously isolate and amplify individual clones

Question 12

What are DNA ligases?

Answer 12

Repair strand breaks in nucleic acid by catalysing formation of phosphodiester bonds
Require ATP or NAD+ as co-factors

Most efficient for joining sticky ends (typical protocol: 20 minutes at room temperature)
Can also ligate blunt ends (but much less efficient, need more enzyme, longer incubation, lower temperatures)

Question 13

Describe the PCR?

Answer 13

Polymerase chain reaction:
PCR selectively amplifies a stretch of DNA from a larger piece of DNA
This is a cyclical process and every 4th and subsequent cycles: the amount of product doubles every cycle we get exponential amplification of the region of interest

Requires a forward and reverse primer which delimit the region of interest
Forward primer - 5’ end of region of interest on coding strand
Reverse primer - 5’ end of region of interest on anti-coding strand

Question 14

What are the stages of PCR?

Answer 14

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 15

What should be considered with PCR?

Answer 15

Primer sequence should be long enough to anneal only with the ends of the region of interest
If they are too short then there is a chance of annealing with other regions of the template

The primers do not have to be perfectly complementary to the annealing site (but the 3’ end that the polymerase recognises should be perfect) – can use to introduce mutations

Question 16

How is cDNA constructed?

Answer 16

Reverse transcriptase, along with poly(T) primer and dNTPs form and RNA-DNA hybrid from mRNA
mRNA template is removed using RNase H or treatment with alkali
3’ end of DNA loops back serves as primer for second strand synthesis by DNA pol
S1 nuclease degrades ssDNA in loop
= cDNA

Question 17

What is the benefit of recombinant protein expression?

Answer 17

You can produce large amounts of protein
You can fuse the protein to a tag: affinity tags, solubility tags, fluorescent tags and epitope tags
You can engineer mutations into protein sequences, so you can investigate protein function

Question 18

What are DNA libraries?

Answer 18

Collections of cloned DNA
They allow us to identify and selectively amplify particular portions of a larger piece of DNA
For example the human genome

Question 19

How do you create a DNA library?

Answer 19

1. The high molecular weight genomic DNA is and the vector are digested with a restriction enzyme
2. The fragments are then fractionated or separated by gel electrophoresis to obtain fragments of a manageable size.
3. The fragments (inserts) are ligated into the digested vector
4. The recombinant vectors are introduced into a host and amplified

Question 20

What is the bluescript plasmid?

Answer 20

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

So if the vector contains an insert = LacZ gene disrupted
Therefore colonies containing a genomic insert = white

Question 21

How do we screen a DNA library?

Answer 21

We screen colonies for presence of gene of interest by replica plating followed by in situ hybridisation (southern blotting)

Colonies are grown on a plate and velvet is pressed into it and transfers them to nitrocellulose filter
The DNA becomes bound to the filter after NaOH treatment and drying
A radioactive probe will hybridise with complementary DNA (gene of interest)
An autoradiograph is used to compare with the master plate - dark colonies = gene of interest

Question 22

What is site-directed mutagenesis?

Answer 22

It alters a gene’s nucleotide sequence - in order to mimic the natural process of evolution

This leads to:
Transgenic organisms e.g. transgenic plants have been engineered for better nutrition - rice
Gene therapy - genetic defects can be corrected e.g. SCID

Question 23

How are nucleic acids fractionated?

Answer 23

Using chromatography, electrophoresis and southern blotting to identify