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
Arthrobacter luteus
Bacillus amyloiquefaciens H
Enzyme
Alu I
Bam HI
Recognition Site
AG CT
G GATCC
Ends produced
Blunt
Sticky
Escherichia coli
Haemophilus gallinarum
Haemophilus infulenzae
Providencia stuartii 164
Eco
Hga
RI
I
Hind III
Pst I
G AATTC Sticky
GACGC(N)
5
Sticky
A AGCTT
CTGCA G
Sticky
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


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
32
P, often by removal of the 5' phosphate of the probe with alkaline phosphatase, and replacement with a radiolabelled phosphate using α -[
32
P]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).
 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
32
P]ATP
Or at the 3' end: add a homopolymeric tail using terminal tranferase and [α
32
P]dATP (or another
32
P-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 modified
Specific modification
G
A + G
C + T
Methylation of base with dimethyl sulfate at pH 8.0.
Makes base susceptible to cleavage by alkali
Treatment with piperidine formate at pH 2.0.
Results in removal of purine bases
Hydrazine opens pyrimidine rings and causes their removal from DNA
C
A > 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.

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)