Hybridisation Techniques

Be able to describe the conditions under which double-stranded nucleic acid molecules are denatured and renatured.

As I believe I've mentioned in a previous post (though I can't be bothered trying to find out which one right now), high temperatures and high pH both cause DNA to become denatured. High temperatures denature DNA by breaking the relatively weak hydrogen bonds between bases of opposing strands, whereas high pH deprotonates some of the bases, leaving fewer places where hydrogen bonds can form. DNA will renature if the conditions are reversed- for example, when temperatures or pH are lowered.

Know hybridisation can occur between complementary DNA and/or RNA molecules. 

When temperatures or pH are lowered, not only can two complementary DNA strands anneal to each other, but RNA can anneal to the DNA as well.

Be able to describe the difference between heterologous and homologous probes. 

Homologous probes are used to find identical genes, whereas heterologous probes are used to find similar genes.

Be able to describe the process of random priming.

Random priming is a technique used to label DNA molecules. The DNA is placed in a solution containing nucleotides labelled in some way- maybe they are radioactive or they have a chemical tracer attached to them. The DNA is then denatured and short primers are added on to the strand. DNA polymerase then creates a complementary DNA strand, using the labelled nucleotides to do so.

Be able to describe the Southern, Northern, and in situ hybridisation techniques and give examples of the uses of these techniques.

Southern blotting is a technique used to find out if identical or related genes are present within chromosomes. Firstly, the DNA to be studied is cut with restriction enzymes and run through agarose gel electrophoresis, a process described in my previous post. This is then blotted onto nitrocellulose paper. The DNA doesn't show up visibly on the nitrocellulose paper, so the paper is placed into a plastic bag along with labelled probes- sequences complementary to the sequence that is being searched for. The colour change or radioactivity produced by the labelled probe then aids in the identification of related sequences.

Northern blotting is a similar technique, but it is used to find out if genes are being expressed. To determine if a gene is actually expressed, Southern blotting is performed, but mRNA is used instead of DNA, as the presence of mRNA is usually a good indication that the gene is being expressed.

In situ hybridisation is a technique to determine where transcripts can be found within tissues or cells. A coloured probe binds to mRNA within the cells. This technique can be used to examine gene expression throughout development.

There is another hybridisation technique called fluorescent in situ hybridisation. In contrast to regular in situ hybridisation, fluorescent in situ hybridisation requires breaking open the cells and nuclei so that the chromosomes can be spread out. These chromosomes can then be denatured and fluorescent probes can be used to determine the location of particular genes on chromosomes.