Electrophoresis

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Electrophoresis

It is used to separate DNA fragments based on their size.

It is accurate enough to be able to separate fragments that are different by only one base in length.

It is widely used in gene technology to separate DNA fragments for identification and analysis.

The technique uses a gel 'plate' or slab, containing agarose, which is then covered in a buffer solution.

Electrodes are added at each end so that a current can be passed through it.

The separation of strands of different lengths occurs because longer strands of DNA get caught up in the agarose gel and are slowed, whereas shorter strands can move more quickly through the gel

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Procedure

It is used to separate DNA fragments based on their size.

It is accurate enough to be able to separate fragments that are different by only one base in length.

It is widely used in gene technology to separate DNA fragments for identification and analysis.

The technique uses a gel 'plate' or slab, containing agarose, which is then covered in a buffer solution.

Electrodes are added at each end so that a current can be passed through it.

The separation of strands of different lengths occurs because longer strands of DNA get caught up in the agarose gel and are slowed, whereas shorter strands can move more quickly through the gel

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Blotting

The fragments can be lifted from the gel for further analysis using Southern Blotting.

A nylon or nitrocellulose sheet is placed over the gel, covered in paper towels, pressed down and left over night.

The DNA fragments are transferred to the sheet and can now be analysed.

DNA fragments are not visible on the sheet.

There are several methods available for showing up for the separated strands.

The simplest is to label the DNA with a radioactive marker before the samples are run.

Placing photographic film over the nitrocellulose sheet shoes the position of DNA samples in the finished gel.

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Using probes

If one particular fragment or sequence of DNA is being searched for, for example a particular gene, then a radioactive DNA probe can be used to check for the presence of that particular sequence

Probe is a short single-stranded piece of DNA (50-60 nucleotides) that is complementary to a section of the DNA being investigated.

The probe is labelled;

Using a radioactive marker (usually ³²P in the phosphoryl groups forming the strand) so that the location can be revealed by exposure to photographic film.

Using a fluorescent marker that emits a colour on exposure to UV light. Fluorescent markers are also used in automated DNA sequencing.

Copies of the probe can be added to any sample of DNA fragments and, because they are single-stranded, they will bind to any fragment where a complementary base sequence is present

Binding by complementary base pairing is annealing.

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Probes continued...

Useful in locating specific sequences;

A specific desired gene that is wanted for genetic engineering

To identify the same gene on a variety of different genomes, from separate species, when conducting genome comparison studies.

To identify the presence or absence of an allele for a particular genetic disease.

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Probes and disease diagnosis

Diagnosis of some genetic diseases and identification of carriers can be made by analysisng DNA using DNA probes.

Probes are made complementary to sequences found in faulty alleles of particular genes.

Scientists are able to place a number of different probes on a fixed surface known as DNA microarray.

Applying the DNA sample to the surface can reveal the presence of faulty or mutalated alleles that match the fixed probes because the sample DNA will anneal to any complementary fixed probes.

In order to anneal, the sample DNA must be broken up into smaller fragments. 

It may also be amplified using PCR so that many copies of each fragment are present.

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