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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.