? Presence of restricfion fragments of DNA in a sample
Involves separation, transfer and hybrdization
Single gene/ larger piece of DNA.
1) DNA is digested with a RE, separated by gel electrophoresis.
2) Transferred to a membrane. Same pattern.
3) The blot is incubated with many copies of a probe. This probe will form base pairs with its complementary DNA sequence and bind to form a ds DNA molecule. Radioactive/ enzyme bound (ALP or horseradish per).
4) The location of the probe is revealed by incubating it with a substrate = colored product.
Nitrocellulose/Nylon paper laid over the gel
The gel is supported on a layer of sponge in a bath of alkali solution
Buffer is sucked through the gel and the nitrocellulose paper
As the buffer is sucked through, it denatures the DNA and transfers the single-stranded fragments from the gel to the surface of the nitrocellulose sheet. Adhere.
The nitrocellulose sheet is peeled off the gel.
The sheet containing the bound ss DNA fragments is placed in a sealed plastic bag together with buffer containing a probe specific for the required DNA sequence. Exposure.
The sheet is removed from the bag and washed. Only probe molecules that have hybridized to the DNA on the paper remain attached.
Autoradiography = the DNA that has hybridized to the labeled probe will show up as bands on the autoradiograph.
Transfer of the DNA to a Membrane
The DNA is denatured –
Transferred onto a membrane.
Incubated with a soln
containing a probe. Comp.
A wash step removes any
probe which is not tightly
bound to the DNA on the
Only DNA that matches
exactly will remain bound
RNA molecules of varying lengths are separated by size and blotted onto nitrocellulose.
A DNA probe (often a cDNA) is then used to identify bands that contain particular sequences.
Useful for determining the conditions under which specific genes are being expressed, including which tissues express which genes ?
Proteins are separated by electrophoresis and blotted onto an appropriate support matrix.
The matrix is then exposed to an antibody to the desired protein and all unbound antibody is washed off.
The bands are detected by the binding of a second antibody that is radioactively labeled and specific for the first antibody.