|
Finding
the piece of DNA containing the gene you want
The
fragments of DNA you have obtained from cutting up the genome
or making cDNA will now be of different types and sizes.
Some
may code for the gene you require others will contain nothing,
half or quarter of a gene in the case of the genomic library.
How
do you sort the interesting from the useless bits: -
1.
The fragments can be separated out using
Gel
Electrophoresis.
2.
The required fragment or band can then be identified by using:
-
A.
A Gene probe
The
probe is a length of single - stranded DNA containing the
complementary base sequence to the gene you require. The DNA
making up the probe is usually labelled in some way and is
made artficially using radioactive isotopes of Phosphorus
32 P in its sugar backbone.
The
gel containing the DNA fragments will then be washed in NaOH
solution; - which breaks the double stranded DNA apart into
single strands for the probe to stick to.
A
nitrocellulose sheet is then placed on top of the gel and
the single strands stick to the sheet in the same positions
as on the original gel. This is called SOUTHERN BLOTTING.
The
probe is then incubated with the nitrocellulose sheet and
its complementary base pairs will stick to the fragment that
contains the gene.
X-ray
film will then be laid over the nitrocellulose sheet and it
will darken where the radioactive probe has stuck , identifying
the band on the gel with your gene.
B.
Guessing the size
If
you know the product of your gene and have an idea of the
number of amino-acids and hence base pairs coding for the
protein, you can select the fragment from the gel based on
its size in Kbp.
The
smallest fragments will be at the bottom of the gel and the
largest pieces at the top. Running size markers with your
original gel is usually necessary for this method to work.
3.
You can now cut out the identified fragment from your gel.
Rinse out the DNA and make lots of copies of your DNA using
the PCR
technique.
You
need as many copies of your gene as possible to ensure that
it will be inserted into a Vector
for the next stage in genetic engineering.
| 
