Biotechnology

Question 1

Biotechnology

Answer 1

The use of organisms, parts of organisms or their products for practical purposes.

Question 2

Recombinant DNA technology often involves: (Overview of the major steps involved)

Answer 2

1. extracting DNA from cells (eg. using detergent and cold ethanol)
2. copying of DNA (eg. using polymerase chain reaction (PCR))
3. cutting DNA into smaller fragments (eg. using restriction enzymes)
4. isolating DNA via separating and locating DNA (eg. using gel electrophoresis to separate fragments by size and using probes to locate useful fragments in the gel).
5. inserting novel DNA into a vector (eg. using a recombinant plasmid or virus) and joining DNA (eg. using DNA ligase enzymes). This molecule containing old and new DNA is now called a recombinant DNA molecule.
6. transforming a cell ( eg. bacterial transformation) where the vector (a recombinant DNA plasmid or virus) enters and transforms a new cell / organism (eg. via electroporation- applying a brief electrical shock or by using thermal shock therapy- rapidly changing temperature).
7. amplification (or cloning) of the recombinant DNA molecule occurs within the transformed cell (eg. a transformed bacterium) when it replicates. The new transgenic organism or a protein product made by it (eg. insulin) are then used for a practical purpose.

Question 3

Polymerase Chain Reaction (PCR)

Answer 3

A technique used for the purpose of copying (amplifying) very small quantities of DNA for analysis

There are three major steps involved that are repeated up to 50 times:

1. Denaturation of original DNA (H bonds between complimentary N bases break at 94°C)
2. Annealing of Primer to DNA (primers bind to complimentary DNA template sequence at 50-60°C)
3. Elongation / Extension of DNA (Taq polymerase enzymes add free DNA nucleotides in a 5’ to 3’ direction to form double stranded DNA at 72° C).

Question 4

Cutting and Joining DNA using Enzymes

Answer 4

1. Restriction endonucleases
   cut DNA at specific recognition sites (enzymes that act like scissors).

Restriction endonucleases (enzymes) are naturally found in bacteria and they cut DNA at specific base sequences called recognition sites. There are two major groups of restriction enzymes: sticky-end restriction enzymes that leave fragments with overhanging ends and blunt-end restriction enzymes that leave clean cut ends.

2. DNA ligase
   bind DNA fragments together (enzymes that act like glue).

DNA ligases are involved in ligation and create phosphodiester bonds between DNA fragments that have been cut previously using the same restriction enzyme.

Question 5

Separating & Isolating DNA fragments using Gel Electrophoresis.

Answer 5

Gel Electrophoresis is a technique used to separate DNA fragments (or proteins) of different sizes.

The DNA fragments made by digestion are of a range of different sizes and are negatively charged so will move through a gel from wells placed near the negative (black) terminal of the tank towards the positive (red) terminal. Smaller fragments move faster (further) through the gel. The size of DNA fragments can then be determined by comparing bands produced in lanes below the wells to bands of known size produced in the ladder lane.

Question 6

Transformation of organisms

Answer 6

Normally recombinant vectors (such as plasmids or viruses containing recombinant nucleic acids) are used to transfer novel genes into ‘host’ cell. In bacteria transformation occurs by using techniques such as electroporation or thermal shock.

Plasmids are self replicating loops of DNA found in bacteria which are easily shared between bacteria. Recombinant plasmids can be easily used to transfer novel genes to both bacteria and also plants

Question 7

Amplification (Cloning) of Recombinant DNA molecules.

Answer 7

Recombinant DNA (rDNA) and genes can be amplified or cloned in a number of ways. One way is by using this rDNA (eg. a plasmid containing a gene of interest) to transform either a bacterial or yeast cell and then when these transformed cells divide by binary fission they also copy this rDNA. These transformed cells are grown in nutrient media (eg. on agar plates or in nutrient broths in bioreactors). Bioreactors are used to culture on a large scale genetically modified microorganisms to extract useful protein products eg. insulin, antibiotics, antigens/ sub-unit vaccines (image to the right shows a bioreactor used to produce antibodies)