Locating and sequencing gene

Specific genes can be located using probes that bind to that particular sequence of DNA.

Probes can be either:

• Fluorescent - detected using UV light.
• Radioactive - detected using x-ray film.

Process:

• DNA probes are short strands of DNA that have a specific base sequence complementary to base sequence of the gene we want to find.
• These probes have a fluorescent or radioactive marker attached.
• The original DNA strand is separated at high temperatures.
• The separated strands are then mixed with the labelled probes.
• The probes will bind to the complimentary base sequence of the target DNA strand if present.
• This can be identified under UV light or X-ray film.

Once genes have been located - strands of DNA can be separated by length to find their position relative to length of DNA/sequence.

Gel electrophoresis separates DNA fragments/strands so smaller strands travel further.

• DNA sample is extracted and isolated e.g. blood or hair etc.
• Restriction endonulceases are used to cut the DNA sample in fragments.
• Fragments multiplied using PCR.
• DNA fragments applied to a gel and voltage is applied.
• Fragments separated by electrophoresis.
• Fragments then transferred to nylon membrane through Southern Blotting.
• DNA probes (radioactive or fluorescent) are attached to core sequences/tandem repeats.
• Membrane placed under x-ray film or UV light to expose relative positions of fragments.

Fragments of DNA can be sequenced to determine the order of bases in a gene. This done using the chain termination method (the Sanger method):

• A mixture is prepared containing a single strand of DNA (the template), DNA polymerase (to join nucleotides together), DNA primers (which initiate the new DNA strand) and free nucleotides (complimentary to the DNA strand). Mixture is put into 4 different tubes.
• Sequencing is achieved by use of terminator bases - modified nucleotides that stop the DNA chain meaning it can no longer extend.
• Each of the four test tubes will have a different terminator base added to it (i.e. A, C, G or T), so the incorporation and joining of a terminator base to the DNA strand is at random.
• Each of the resulting fragments will be of different lengths due to random joining of terminator bases, but all will end in the same base.
• The terminator bases are labelled with a fluorescent or radioactive marker so can be identified under UV light or X-ray film.

Gel electrophoresis is then used to separate out the strands and identify their relative position so by reading from bottom to top - the DNA sequence can be built up one base at a time.

Many genes are too long to be sequenced using the Sanger method all in one go.

Therefore they must be cut up using restriction enzymes, with the smaller fragments sequenced.

These smaller sequences must be pieced back together so that the whole sequence can be read. This is done using restriction mapping:

• Different restriction enzymes are used to cut labelled DNA into fragments.
• DNA fragments separated via electrophoresis.
• Size of fragments are used to determine the relative locations of the cut sites
• A restriction map of the DNA is produced - a diagram of the piece of DNA showing the different cut sites - recognition sites of the enzymes.