The Molecular Biology Notebook OnlineYou are here: Biotechnology > Introduction > DNA Digestion
Cutting DNA
Dr Chromo is working on a DNA project, and he is cutting the DNA he has just prepared. We might ask
- Why is he doing that?
- What kind of tools are needed to cut DNA?
- What do we do with cut DNA?
- How do we use Biolab to simulate digestion of our DNA
Why cut DNA?
Not many experiments need the use of whole molecules of DNA (One chromatid contains only one molecule of DNA, and that molecule can be several metres long). Most experiments need smaller pieces of DNA. For example, when you want to introduce a fragment of DNA from one organism into the genome of another one, you need to cut out this piece of DNA first. Most of the time you also need to cut the DNA in specific regions, and know exactly where you are in the DNA sequence. With the research tools available today, all this is possible.
Things that cut DNA
1) Physical strength
Very big molecules of DNA can be broken by ultrasound. Big molecules
of DNA make a viscous solution. After sonication
, the
solution loses its viscosity. This is because the DNA has been broken down. Usually
this operation is performed on ice, or in a cold room.
2) Non-specific DNAses
Many enzymes cut DNA. An enzyme that cuts DNA is called a DNAse. An enzyme that cuts RNA is an RNAse. These enzymes cut any DNA (or RNA) anywhere
in the molecule. Some will cut only single stranded DNA (like MungBean
DNAse). These classes of enzymes can prove useful in some experiments.
3) Specific DNAses ( also known as restriction endonucleases or restriction enzymes)
 |
Restriction endonucleases cut DNA at specific sites (sequences of DNA) There are many
enzymes of this type. (more than 400 have been isolated, not all of them are used in research laboratories.)
Each enzyme will recognise a specific sequence
of double stranded DNA and cut at that sequence,
and nowhere else under optimal conditions.
The movie illustrates this concept. |
Some enzymes make a simple cut across both strands of DNA, leaving what are called blunt ends.
Others leave what are called sticky ends, small single stranded DNA overlapping the cut. The hanging DNA can stick to a complementary sequence, and a longer DNA can be formed this way.
Restriction endonucleases are extracted from bacterial cultures and purified before use in genetic engineering. They are bought from pharmaceutical companies and are quite expensive. They are usually kept in a small tube, in a freezer. It is important to keep them refrigerated at all times. It is also very important to wear gloves when handling the tubes, or the solution could become contaminated with other DNA or enzymes.
Now it is all in pieces... What do I do?
Now our DNA has been cut into smaller pieces, we can estimate the size of the fragments, by running an agarose gel. This gel can be used to analyse the DNA (using blotting and hybridization technology).
Digestion of DNA is also an elementary step in gene cloning technology, and in the mapping of genes.
Using Biolab to Digest your DNA
The Molecular Biology Notebook's Virtual Lab, Biolab, has been specially designed to allow you to cut your DNA safely. You have at your disposal a selection of restriction enzymes. You can try them out on the DNA you just loaded on the software.
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Restriction Digest on Wikipedia
Restriction Digest at Answers. com
Related Links
-RESearch - a new computer program for DNA analysisThe main design philosophy behind the development of RESearch was to make the basic DNA analysis too
-Restriction Mapper
Restriction Mapper is a web site that finds restriction endonuclease cleavage sites in DNA sequences
-WebCutter
Webcutter is an on-line tool for restriction mapping nucleotide sequences. Now the new version Cutte