0.1 Basic principles of genetics  (Page 14/19)

Lecture 14. genes can be manipulated by molecular tools i

Progress in any scientific discipline is dependent on the availability of techniques and methods that extend the range and sophistication of experiments which may be performed. Over the last 30 years or so this has been demonstrated in spectacular fashion by the emergence of molecular genetics. This field has grown rapidly to the point where, in many laboratories around the world, it is now routine practice to isolate a specific DNA fragment from the genome of an organism, determine its base sequence, and assess its function. What is particularly striking is that this technology is readily accessible by individual scientists, without the need for large-scale equipment or resources outside the scope of a reasonably well-found research laboratory.

Although there are many diverse and complex techniques involved, the basic principles of genetic manipulation are reasonably simple. The premise on which the technology is based is that genetic information, encoded by DNA and arranged in the form of genes, is a resource which can be manipulated in various ways to achieve certain goals.

DNA extraction. Depending on the cell characteristics, DNA extraction from animal cells differs from DNA extraction from plant or prokaryotic cells. Link to Gentra Puregene Protocols for technical reports on DNA extraction.

Hybridization techniques. Southern blotting, Northern blotting and in situ hybridization (including fluorescent in situ hybridization - FISH). Hybridization techniques allows picking out the gene of interest from the mixture of DNA/RNA sequences. Hybridization only occurs between single stranded and complementary nucleic acids. The level of similarity between the probe and target determines the hybridization temperature. See the overview of blotting techniques from the Biology Hypertextbook, an animation of Southern blotting , and an example of DNA fingerprinting .

Enzymatic modification of DNA. DNA ligase and restriction enzymes (sticky ends, blunt ends). Most restriction enzymes recognize palindromic sequences. These are short sequences which are the same on both strands when read 5' to 3' (such as he MspI restriction site CCGG and that of EcoRI GAATTC). See the action of Eco RI .