INTRODUCTION
The development of recombinant DNA techniques has spawned many new
approaches to the analysis of genes and gene products .
Geneticists can isolate and characterize essentially any gene from any
organism. However, the isolation of genes from large Eukaryotic genomes
is sometimes a long and laborious process .
Once a gene has been cloned its expression can be investigated in even
the most complex organism such as humans.
Let’s consider some of the most important methods used to investigate the
structure of genes(DNA),their transcripts (RNA) and their final products
( usually proteins)
NUCLEIC ACID
HYBRIDIZATION
Nucleic acid hybridization is a Basic technique In molecular biology. Which
takes advantage of the ability of individual single standard nucliec acid
molecules to form double stranded molecules
Nucleic acid hybridization is a process used to identify specific DNA
sequences Specific DNA probess are denatured and annealed to sample
DNA that has also been denatured
Probe – a single-stranded nucleic acid that has been radiolabelled and is
used to identify a complimentary nucleic acid sequence that is membrane
bound
Probes used in hybridization reactions are usually chemically synthesized
DNA or RNA that has been labelled with a fluorescent dye or radioactive
isotope such as 32P
SOUTHERN HYBRIDIZATION
The name of this technique is derived from the name of its inventor – E M Southern
DNA : DNA hybridization forms it’s basis
The following steps describe the Southern transfer procedure.
Digest DNA with the restriction enzyme of choice.
Load the digestion onto a agarose gel and apply an electrical current. DNA is
negatively charged so it migrates toward the “+” pole. The distance a specific
fragment migrates is inversely proportional to the fragment size.
Stain the gel with EtBr, a fluorescent dye which intercalates into the DNA molecule.
The DNA can be visualized with a UV light source to assess the completeness of the
digestion.
Denature the double-stranded fragments by soaking the gel in alkali (>0.4 M NaOH)
Transfer the DNA to a filter membrane (nylon or nitrocellulose) by capillary
action. This process is known as blotting.Typically a Southern transferst
setup contains (from bottom to top):
buffer
sponge
filter paper
the gel containing the nucleic acid
a nylon or nitrocellulose membrane
more filter paper
paper towels to catch the buffer that passed through all of the above
Nitrocellulose membrane is now removed from blotting stack.DNA is
permanently
immobilized on membrace by baking it at 80°C in vacuo.
Prepare a probe by nick translation or random, oligo-primed labelling.
Add the probe to a filter (nylon or nitrocellulose) to which single-stranded
nucleic acids are bound. (The filter is protected with a prehybridization solution
which contains molecules which fill in the spots on the filter where the nucleic
acid has not bound.
Hybridize the single-stranded probe to the filter-bound nucleic acid for 24 hr.
The probe will bind to complementary sequences.
Wash the filter to remove non-specifically bound probe.
Expose the filter and determine whether binding occured
NORTHERN HYBRIDIZATION
In this technique RNA bands are separated by gel electrophoresis and the
RNA bands are transferred onto a suitable membrane eg:
diazobenzyloxymethyl(DBM)paper or nylon membrane
The bands are hybridized with radioactive single stranded DNA probe and
the bands showing hybridization are detected by auto radio graphy.
extension of Southern blotting technique
DIFFERENCE
