Download Recombinant DNA Technology

DNA technology
Determining the molecular sequence of DNA that makes up the genome of different
organisms is an international scientific goal, several laboratories are participating
worldwide in this task
Recombinant DNA Technology
Techniques for
- Isolation
- Digestion
- Fractionation
- Purification of the TARGET fragment
- Cloning into vectors
- Transformation of host cell and selection
- Replication
- Analysis
- Expression of DNA
How do we obtain DNA and how do we manipulate DNA?
Quite straightforward to isolate DNA
For instance, to isolate genomic DNA
1. Remove tissue from organism
2. Homogenise in lysis buffer containing guanidine thiocyanate (denatures
proteins)
3. Mix with phenol/chloroform - removes proteins
4. Keep aqueous phase (contains DNA)
5. Add alcohol (ethanol or isopropanol) to precipitate DNA from solution
6. Collect DNA pellet by centrifugation
7. Dry DNA pellet and resuspend in buffer
8. Store at 4°C
Enzymes that can cut (hydrolyse) DNA duplex at specific sites. Current DNA
technology is totally dependent on restriction enzymes.
Restriction enzymes are endonucleases
Restriction enzymes recognise a specific short nucleotide sequence
This is known as a Restriction Site
The phosphodiester bond is cleaved between specific bases, one on each DNA
strand
Examples of restriction enzymes and the sequences they cleave
Source microorganism
Enzyme
Arthrobacter luteus
Alu I
Bacillus amyloiquefaciens H
Recognition Site
Ends produced
AG CT
Blunt
Bam HI
G GATCC
Sticky
Escherichia coli
Eco RI
G AATTC
Sticky
Haemophilus gallinarum
Hga I
GACGC(N)5
Sticky
Haemophilus infulenzae
Hind III
A AGCTT
Sticky
Providencia stuartii 164
Pst I
CTGCA G
Sticky
Nocardia otitiscaviaruns
Not I
GC GGCCGC Sticky
DNA fractionation
Separation of DNA fragments in order to isolate and analyse DNA cut by
restriction enzymes
Electrophoresis
Linear DNA fragments of different sizes are resolved according to their size
through gels made of polymeric materials such as polyacrylamide and agarose.
For instance, agarose is a polysaccharide derived from seaweed - and gels formed
from between 0.5% to 2% (mass/volume i.e. 0.5 to 2.0g agarose/100 ml of
aqueous buffer) can be used to separate (resolve) most sizes of DNA
DNA is electrophoresed through the agarose gel from the cathode (negative) to
the anode (positive) when a voltage is applied, due to the net negative charge
carried on DNA
When the DNA has been electrophoresed, the gel is stained in a solution
containing the chemical ethidium bromide. This compound binds tightly to DNA
(DNA chelator) and fluoresces strongly under UV light - allowing the
visualisation and detection of the DNA.
Analysing complex nucleic acid mixtures (DNA or RNA)
The total cellular DNA of an organism (genome) or the cellular content of RNA
are complex mixtures of different nucleic acid sequences. Restriction digest of a
complex genome can generate millions of specific restriction fragments and there
can be several fragments of exactly the same size which will not be separated
from each other by electrophoresis.
Techniques have been devised to identify specific nucleic acids in these complex
mixtures
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Southern blotting - DNA
Northern blotting - RNA
These techniques are not to be confused with Western blotting, which is used to
analyse PROTEINS which have been immobilised on nitrocellulose/nylon filters.
Proteins which have been separated by polyacrylamide gel electrophoresis
(PAGE) are transferred to nitrocellulose/nylon filters and the filter is probed with
antibodies to detect the specific protein - similar to the method used for
expression library screening.
Southern blotting
This technique, devised by Ed Southern in 1975, is a commonly used method for
the identification of DNA fragments that are complementary to a know DNA
sequence. Southern hybridisation, also called Southern blotting, allows a
comparison between the genome of a particular organism and that of an available
gene or gene fragment (the probe). It can tell us whether an organism contains a
particular gene, and provide information about the organisation and restriction
map of that gene.
In Southern blotting, chromosomal DNA is isolated from the organism of
interest, and digested to completion with a restriction endonuclease enzyme. The
restriction fragments are then subjected to electrophoresis on an agarose gel,
which separates the fragments on the basis of size.
DNA fragments in the gel are denatured (i.e. separated into single strands) using
an alkaline solution. The next step is to transfer fragments from the gel onto
nitrocellulose filter or nylon membrane. This can be performed by
electrotransfer (electrophoresing the DNA out of the gel and onto a
nitrocellulose filter), but is more typically performed by simple capillary action.
In this system, the denatured gel is placed onto sheet(s) of moist filter paper and
immersed in a buffer reservoir. A nitrocellulose membrane is laid over the gel,
and a number of dry filter papers are placed on top of the membrane. By capillary
action, buffer moves up through the gel, drawn by the dry filter paper. It carries
the single-stranded DNA with it, and when the DNA reaches the nitrocellulose it
binds to it and is immobilised in the same position relative to where it had
migrated in the gel.
The DNA is bound irreversibly to the filter/membrane by baking at high
temperature (nitrocellulose) or cross-linking through exposure to UV light
(nylon).
The final step is to immerse the membrane in a solution containing the probe either a DNA (cDNA clone, genomic fragment, oligonucleotide) or RNA probe
can be used. This is DNA hybridisation - in other words the target DNA and the
probe DNA/RNA form a 'hybrid' because they are complementary sequences and
so can bind to each other. The probe is usually radioactively labelled with 32P,
often by removal of the 5' phosphate of the probe with alkaline phosphatase, and
replacement with a radiolabelled phosphate using α -[32P]ATP and polynucleotide
kinase. The membrane is washed to remove non-specifically bound probe (see
washing & stringency conditions), and is then exposed to X-ray film - a process
called autoradiography. At positions where the probe is bound, α -emissions
from the probe cause the X-ray film to blacken. This allows the identification of
the sizes and the number of fragments of chromosomal genes with strong
similarity to the gene or gene fragment used as a probe.
The principle of Southern blotting
What Southern blotting can tell us
1. Whether a particular gene is present and how many copies are present in
the genome of an organism
2. The degree of similarity between the chromosomal gene and the probe
sequence
3. Whether recognition sites for particular restriction endonucleases are
present in the gene. By performing the digestion with different
endonucleases, or with combinations of endonucleases, it is possible to
obtain a restriction map of the gene i.e. an idea of the restriction enzyme
sites in and around the gene- which will assist in attempts to clone the
gene.
4. Whether re-arrangements have occurred during the cloning process
Northern blotting
Northern blotting is a simple extension of Southern blotting - and derives its
name from the earlier technique. It is used to detect cellular RNA rather than
DNA. Initially, it was thought that RNA would not bind efficiently to
nitrocellulose, and other modified materials were synthesised for use as a
membrane. However, it was then shown that when RNA was denatured, that it
would also bind efficiently to nitrocellulose. This means that the RNA has to be
unfolded into a linear strand before it will bind efficiently to nitrocellulose.
Chemicals such as formaldehyde and methylmercuric hydroxide can be used to
denature the RNA - breaking down hydrogen bonding structure in the molecule.
Alkali is not used to denature the RNA - since RNA is degraded under alkaline
conditions.
Isolating RNA
RNA is extracted from the cells of interest - but precautions must be taken to
avoid degradation of the single-stranded RNA by ribonuclease (RNase), which is
found on the skin and on glassware. Wear gloves, use specially treated plastics
and glassware to avoid accidently introducing ribonuclease to extraction prep.
Addition of diethylpyrocabonate (DEPC) inhibits ribonuclease activity and
baking at high temperature destroys ribonuclease activity (only useful for treating
heat resistant equipment, such as glassware).
DNA sequencing: Maxam & Gilbert sequencing
For this method, need to use DNA fragments ~500 nucleotides - for instance by isolating
restriction fragments of the DNA that is to be sequenced. The method is reliable for
sequencing up to ~250-300 nucleotides at a time. The technique requires that the target
DNA is end-labeled (usually radioactively).
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Either at the 5' end: add alkaline phosphatase to remove 5' phosphate, and
polynucleotide kinase to add back a radiolabeled phosphate to the 5' OH group
using [α 32P]ATP
Or at the 3' end: add a homopolymeric tail using terminal tranferase and [α
32
P]dATP (or another 32P-labeled deoxyribonucleotide triphosphate)
Both single-stranded (ss) and double-stranded DNA (ds) can be sequenced. If ds DNA is
used, then the label must be removed from one end, so that fragment sizes can be gauged
by the distance to the end of the molecule from one unique label at the other end.
The M&G method involves the chemical degradation of DNA
The process requires addition of chemicals that bring about cleavage of DNA at specific
positions. (The Sanger method involves DNA synthesis).
Either 4 or 5 separate chemical reactions are performed. The reactions are carried
out in two stages:
Stage 1: Specific chemical modification of bases in the DNA
Stage 2: Chemical cleavage of sugar-phosphate backbone at modification site
Stage 1: Specific chemical modification of bases in the DNA
Base
Specific modification
modified
G
Methylation of base with dimethyl sulfate at pH 8.0.
Makes base susceptible to cleavage by alkali
A+G
Treatment with piperidine formate at pH 2.0.
Results in removal of purine bases
C+T
Hydrazine opens pyrimidine rings and causes their removal from DNA
C
In high ionic strength (1.5 M NaCl) only cytosine reacts with hydrazine
Treatment with 1.2 M NaOH at high temperature (90°C) gives strong
cleavage at A, less at C
Stage 2
When bases are modified and destroyed by the treatments in stage 1, piperidine at 90°C is
used to cleave the sugar-phosphate backbone at the site.
A>C
For example:
The 'trick' in the reaction is to limit incubation times with base-modifying reagents
and/or the concentrations of reagents used, so that a ladder of progressively longer
molecules is generated in the M&G sequencing reactions.
The differently sized/labelled fragments are separated by polyacrylamide gel
electorphoresis (PAGE)