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A Beginners' Guide to Molecular Biology



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Transfer of nucleotides from gel to membrane

We started with a sample of an organism. With great effort, we extracted the DNA from it and digested it with restrictionask Dr Chromo! enzymes. Then we separated the fragments into different sized bands on an agaroseask Dr Chromo! gel by electrophoresisask Dr Chromo!. Now we have our finished gel, we can cut out a band, and purify the DNA contained in it. This DNA fragment might be good enough to be cloned in a plasmidask Dr Chromo! and transferred into a bacterium which make numerous copies of it (clone it) (for future experiments, and use in medicine, for example). This fragment could also be used as a probeask Dr Chromo! to analyse DNA from other sources.

Blotting DNA onto a membrane

A copy of the DNA on the gel can be made on a nylon or nitrocellulose membrane. The membrane is highly absorbant to DNA (and lipids). The process is known as blotting and it enables a copy of the gel to be made that will last almost indefinilty. Blotting is performed not only to make a record, but also because the DNA on the gel will diffuse out of position over time.

Blotting is most frequently done on nitrocellulose membranes which are quite sturdy and can be labeled with a pen to help record keeping. There are several techniques to perform this transfer of the DNA from a gel to a membrane. They involve simple principle of physics.

Capillarityask Dr Chromo! blotting

A paper towel blot is one of the methods used. The bufferask Dr Chromo! migrates by capillaryask Dr Chromo! action through the gel and the membrane and into the paper towels. DNA is lifted off the gel and sticks to the membrane.

After 18 hours under pressure (we usually do this overnight) the gel is discarded as it does not contain DNA any more. The nitro-cellulose membrane has the DNA on it's surface. DNA is fixed on the membrane either with UV rays or with heat. When dry, the membrane can be kept at room temperature.

Vacuum blot

In a vacuum blot, instead of using capillary forces, the force that will transfer the DNA is a vacuum.


Vacuum blotting can be performed in two hours instead of 18. It is therefore a much faster method of transferring DNA. The membrane can then be used in hybridisation experiments.

Hybridization experiments
The principle behind the method of hybridisation of DNA is very simple. DNA is double-stranded, and under some conditions, the two strands can separate. When the DNA is single-stranded, a strand can hybridise with its own complementary strand under optimal conditions, or, hybridise with whatever molecule of DNA thas a complementary sequence of bases.


The membrane we just prepared is covered in double-stranded DNA. The DNA is not visible, that is why the membrane is white. By immersing it in dilute sodium hydroxide (NaOH), DNA is melted melted, and we then have a membrane with single-stranded DNA on it. The membrane is placed in a special cylinder, or bottle. (A)

There are methods that enable us to label DNA fragments so that they can be seen in certain conditions. One of them is radioactivityask Dr Chromo!. We prepare a DNA fragment and label it with radioactivity - this is now called a 'probe'. We then separate the 2 helices so the DNA probe becomes single-stranded. We pour a solution containing this labelled DNA (B) into the bottle containing the membrane. The membrane is incubated with the probe for some time (as long as 18 hours), and then is rinsed several times (D). The probe has hybridised with our DNA in the places it found a DNA sequence which would complement it, making it radioactive. These bands of radioactively labelled DNA can be visualised using a photographic film.



Applications

Blotting and hybridising experimentscan be performed safely in front of your PC using Biolab.

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