1. Electrophoresis and Blots

2. Principles recap
Nucleic acids (DNA, RNA) move from the negative to the positive pole during gel electrophoresis
The resistance to movement is due to the sieving effect of the gel matrix
The rate of migration is related to the size of the nucleic acid

3. Agarose versus acrylamide
Acrylamide is a crosslinked synthetic polymer useful in separating DNA molecules of a few hundred bases in length
The crosslinking reaction is carried out within the molding apparatus
The final gel is transparent and usually used in a vertical apparatus
Agarose is a polysaccharide similar to starch useful in separating DNA and RNA molecules of a few hundred to tens of thousands on bases long
A gel is created by boiling agarose into solution and allowing it to cool within a molding apparatus
The final gel is translucent and usually used in a horizontal apparatus

4. Restriction enzymes I Origin and native utility
Some restriction enzymes are used by bacteria to protect themselves from invading DNA
A “restriction-modification” system exists that marks bacterial DNA by methylating it on certain sequences
A restriction enzyme specific for those sequences cannot digest DNA if it is methylated
Invading viral DNA is unmarked and subject to cleavage at those sequences

5. Enzymes Restriction enzymes Generally bacterial in origin
But may come from lower eukaryotes, viruses or transposable elements
Cleave DNA at short palindrome sites
The palindromes may be 2 or more nucleotides long
Unusual forms may recognize sequences up to 30 bases long
These are used in site specific recombination events
Cleavage usually results in a 5’ phosphate and a 3’ hydroxyl
The resulting ends may be single stranded over a short region, or double stranded (blunt ends)
The cleavage sites may be covalently linked back together by DNA ligase
Enzymes

6. Restriction enzymes III
A restriction enzyme with a 6 base recognition site would produce on average a fragment size of 4096 bases in length when used to cut single copy DNA
The average fragment size can be determined for any recognition site by the formula 4n where n is the number of bases in the recognition site
Size of recognition sequence
Average size of fragment 4
256 5
1024 6
4096 8
65536

7. Agarose gel electrophoresis
Gels are loaded with samples of restricted DNA and electrophoresed for minutes to hours
The voltage is turned off and the gel is stained with the intercalator ethidium bromide
DNA may be visualized in the gel by illuminating it with ultraviolet light
Linear relationship between mobility and the logarithm of the DNA size

8. Size determination
Size markers are commonly used in electrophoresis experiments
These are duplex DNA fragments of a known size
The migration distance of a fragment may be compared to the migration of the size markers directly on the gel
Alternatively the migration of a fragment may be determined by interpolation from a plot of the distance migrated by the size markers
The distance migrated is related to the inverse log of the molecular weight of the DNA fragment

9. Digesting human chromosomal DNA
produces a range of fragment sizes due to cleavage of single copy DNA
Three billion base pairs cleaved by a restriction enzyme recognizing 6 bases results in 750,000 fragments
They appear as a background smear of DNA cleaved into all possible sizes by the restriction enzyme
The fragments are of random size with an average length of >4096 bases
Repetitive sequence can increase the average size, because usually a given restriction site will not be found within it
If a restriction site is in a repetitive sequence, digestion will produce a large number of fragments of identical length
This will appear as a band in the background smear of fragments

10. Once an agarose gel of digested chromosomal DNA has been stained, individual DNA sequences may be detected on the gel by hybridization
The DNA within the gel is denatured by exposing it to a solution of sodium hydroxide
The DNA is then neutralized and transferred out of the gel onto a membrane that binds DNA
This is a procedure known as blotting
It exposes the DNA to the surface so that it may hybridize to complementary sequences
The membrane bound DNA is then hybridized to a short specific sequence known as a probe
Southern Blot

11. Probe
A probe is an oligo or poly nucleotide that represents known DNA sequence
It is usually a sequence complementary to a gene
But it only need be complementary to DNA sequence within the chromosomal DNA itself
It is either single stranded to begin with or is denatured to make it single stranded
It is labeled with radioactive phosphorous or a fluorescent adduct

12. Hybridization
The membrane is “prehybridized” with non-specific DNA to block non-specific binding sites on the membrane prior to hybridization
It is then hybridized to the probe
The temperature and salt conditions are controlled to insure optimal hybridization between the probe and the target sequences
The single stranded probe finds its complement in the membrane bound DNA and forms a double helix
Unbound probe is then washed off

13. X-ray film exposure The membrane is exposed to X-ray film
Radioactive probes expose the film whereever they hybridized on the blot due to emission of electrons (beta particles)
Fluorescent probes catalyze a light yielding reaction using energy supplied in a reaction cocktail
Development of the X-ray yields an autoradiogram
Bands on the film locate the chromosomal restriction fragments complementary to the probe DNA

14. Overall Southern Blots
DNA is digested with restriction enzymes and electrophoresed through an agarose gel
The contents of the gel are denatured in situ and then transferred onto a membrane that binds DNA
The DNA is hybridized to radioactive DNA complementary to the gene of interest
Unhybridized DNA is washed away and the membrane exposed to X-ray film
The technique can be adapted to identify any source of DNA containing a gene of interest or to RNA detection (northern blots)
Overall

15. Restriction fragment length polymorphism (RFLP)
Chromosomal DNA from two people differs due to a random variations in sequence
There are millions of single nucleotide polymorphisms between two unrelated individuals
If one of these sequence variations results in the creation or destruction of a restriction site, then the fragment sizes of DNA will differ between these two people
This can be detected by southern blots

16. Example I RFLP point mutation
A gene sequence contains two Eco R1 sites separated by 4000 nucleotides
A patient has suffered a mutation in one of his two homologous chromosomes such that a single nucleotide change has created a new EcoR1 site within the gene sequence
Now two fragments are created of 1500 and 2500 bases in length
The probe is complementary to sequence located within the 2500 base long fragment
Detection by southern blot of these two fragments will result in a 4000 base long fragment in normal DNA from one homologous chromosome and two fragments from the other homolog

17. DNA from normal individual digested with Eco R1 and
electrophoresed through agarose
DNA from mutant individual digested with Eco R1 and electrophoresed through agarose

18. Example II RFLP deletion mutation
How could these two
patterns arise by deletion?

19. Example III Amplification

20. Other blots Northern
Northern blots are mainly used to measure of the amount of a specific RNA in the cell
The blot is prepared and hydridized like a southern blot
There are a few minor technical alterations
The RNA is usually folded into a secondary structure so it must be denatured before and during electrophoresis
Bands on the autoradiogram are a measure of the presence of RNA from a particular gene
The more intense the band, the more RNA on the blot

21. Protein electrophoresis
Proteins, unlike DNA, do not have a constant size to charge ratio
In an electric field, some will move to the positive and some to the negative pole, and some will not move because they are neutral
Native proteins may be put into gel systems and electrophoresed
However native proteins are sometimes difficult to prepare without degradation
An alternative to native protein gels forces all proteins to acquire the same size to charge ratio

22. SDS-polyacrylamide gel electrophoresis
A sample of protein, often freshly isolated and unpurified, is boiled in the detergent sodium dodecyl sulfate and beta-mercaptoethanol
The mercaptoethanol reduces disulfide bonds
The detergent disrupts secondary and tertiary structure
On the molecular level, proteins are stretched out and coated with the detergent (which has a negative charge) by this treatment
They will then migrate through a gel towards the positive pole at a rate proportional to their linear size
Molecular weights with respect to size markers may then be determined

23. Other blots II Western
Western blots measure the amount of a specific protein in a cell
This is a blot of an SDS- polyacrylamide gel onto which cellular protein is applied
The protein is denatured so it will enter the gel as a stretched out random coil
The proteins separate by size
They are blotted onto a membrane
Western blots are probed using antibodies
The antibodies are labeled. Those that remain bound to their specific targets identify specific proteins in the cell
The intensity of the signal reflects the level of expression
It can also reveal mutations by anomalies of migration
A deletion might cause a shorter protein for example