1. Biotechnology

3. Biotechnology
Biotechnology means using biological technology to produce useful organisms such as plants, animals and micro-organisms.
The organisms themselves or the products which they produce may be useful.
To do this we use Gene technology to modify the DNA of these organisms.
We alter genes, remove genes, add extra copies of genes or add genes from other organisms.

4. The Tools of Biotechnology
Genes have 2 main components:
The protein coding region – contains the nucleotide triplet codes which code for specific amino acids and the order they are arranged in. This is a universal code, so in theory the same protein can be made by any organism.
The promoter region – controls gene expression. Regulates in which tissue the gene should be expressed, at what time and in response to what stimulus the gene is transcribed. (only work within Kingdoms.)

5. Collecting the DNA
A piece of tissue or a blood sample contain many cells and can be used to extract DNA.
The process of collecting the DNA has 4 steps.

6. Collecting the DNA
Isolate the DNA from the rest of the cell – cells are mechanically broken open, then using detergents and enzymes, the cell walls and membranes (including nuclear membranes) are broken, thus releasing the DNA.

8. Collecting the DNA
Remove the unwanted cell debris – done either by filtering the extract or centrifuging the mixture. The filtrate will now contain the DNA along with some unwanted proteins.

9. Collecting the DNA
Remove the unwanted protein – done by adding a protease enzyme that breaks down protein.
(In some research labs phenol will then be added to destroy all of the unwanted protein.)

10. Collecting the DNA
Precipitate out the DNA – DNA can now be precipitated out by pouring a layer of ice-cool ethanol over the surface of the filtrate.

11. The Scissors (Restriction Enzymes)
Restriction Endonucleases
These are enzymes which occur naturally in bacteria.
Their function is to protect the bacterial cells from infection by foreign DNA (viral DNA) by cutting the DNA into smaller pieces.
Restriction enzymes cut DNA at specific base sequence recognition sites, usually 4 – 8 base pairs in length.

12. The Scissors (Restriction Enzymes)
The base sequence recognition site is a Palindrome (reads the same in both directions).
5’ G A A T T C 3’
3’ C T T A A G 5’
Some restriction enzymes cut DNA between 2 specific bases at the recognition site resulting in “blunt ends” on the two cut ends of the DNA.

13. Blunt Ends

14. Restriction Enzymes
Other restriction enzymes cut DNA in a staggered manner at the recognition sites, resulting in “sticky ends” which are overhangs on the 2 cut ends of the DNA.

15. Restriction Enzymes
These Sticky ends are able to bind to complementary sticky ends on other DNA.
In this way, segments of DNA can be inserted into or added to other segments of DNA, which have been cut by the same restriction enzymes to produce matching sticky ends.

16. Restriction Enzymes
Restriction enzymes are named from the genus, species and strain of the bacterium from which they were isolated.
E.g. Eco R1 is from E. coli Strain RY13 and 1 = the first restriction enzyme isolated from E.coli.
There are now more than 400 restriction enzymes in use.

17. To Do
Biotech book A pg 10 – 14 Activities 1-3

18. The Glue – Ligase Enzymes
Combining the sticky ends of 2 different DNA strands is only temporary, because only a few H-bonds hold the ends together.
The joins can be made permanent by the use of DNA Ligase Enzymes.
These catalyse the formation of phosphodiester bonds between the phosphates and the sugars on the sides of the DNA ladder during replication and repair.

19. Ligase

20. Cloning
A clone is a vector molecule carrying a unique fragment of DNA.
It can also be used to describe a colony of cells derived from a single cell, which has in turn received a single plasmid molecule.
(plasmids – small circular double stranded pieces of DNA found mostly in bacteria.)

21. DNA Carriers A Vector is a vehicle that carries DNA into a host cell.
The engineered piece of DNA has to be put back into the cell in order to function, as it needs the rest of the cell machinery to function.
Can be done by natural vectors (bacteria, viruses, yeast) or when natural vectors can’t be used (in plant and animal cells) electroporation or a DNA gun can be used.

22. Bacteria as Vectors
The usual vectors used are the plasmids from bacteria.
These are circles of DNA that can be removed from the bacteria and are opened using restriction enzymes to leave sticky ends.
The piece of DNA of interest can be cut with the same restriction enzymes, and inserted into the plasmid.
This can be placed back in the bacteria.

23. Bacteria as Vectors
The bacteria divides, replicating the foreign DNA until you have many copies.
These can now be used in different ways.
To produce many copies of the gene of interest, which could then be isolated and placed into another organism such as a plant.
To produce the protein that the gene of interest codes for, e.g. insulin and growth hormone.
The engineered bacteria may have a new function, such as cleaning up oil spills.

25. Viruses as Vectors
A virus is a string of DNA (RNA in some viruses), that is covered with a protein coat.
Virus that attack bacteria are called Bacteriophages or phage for short.
New DNA can be spliced into the virus DNA, which is then returned to the virus coat.

27. Viruses as Vectors The Phage can then infect a bacteria.
Inside the bacteria the DNA replicates, using the bacterial cell machinery, to make new phage particles.
These in turn infect more bacteria; and so on.
It is possible to carry DNA into animal cells using viruses.

29. Viruses as Vectors
Viruses can be used to clone larger fragments of DNA then plasmids.
They are also often host specific, will only invade certain cells.
E.g. a respiratory virus that invades the lungs has been used experimentally to insert a healthy cystic fibrosis gene into the lungs of rats and the nassal passages of humans.

30. Yeast as a Vector
Sometimes the protein to be made is too complicated for a prokaryote, so a eukaryote cell needs to be used.
Yeast has plasmids so it is ideal.

31. Electroporation
An electric current is used to force DNA across a cell membrane.
This is useful for introducing plasmids into eukaryotic cells.

32. DNA Gun
The DNA of interest is coated onto microscopic pellets (gold or tungsten) and fired into a cell with a gun.
This is useful for introducing DNA into plant cells that are not susceptible to infection.

34. DNA Synthesis
We are now able to synthesise small, one-sided strands of DNA, about 100 bases long, called Oligonucleotides.
Scientists simply type in the sequence they want into a computer joined to the synthesiser, and the machine joins nucleotides together in the right order.

35. DNA Synthesis
Chemically-made small oligonucleotides are usually called Synthetic Oligonucleotides.
They are important for making a primer for PCR.
They can also be used as Gene Probes: if you know the sequence of nucleotides of the gene you want, you can make a complimentary oligonucleotide that will attach to a DNA if there is a perfect match in their sequences.

36. DNA Synthesis
Oligonucleotides are also useful in finding a mutation within a gene where as little as 1 base has changed.
This is important in genetic screening, because a single base change can cause an abnormal protein to be made which can lead to a genetic disease.

37. Polymerase Chain Reaction
This is a method by which a minute piece of DNA can be quickly copied many times in vitro.
The advantage that this method has over cloning is that billions of copies of a piece of DNA can be made in hours as opposed to weeks.

38. Starting Materials
Pieces of double stranded DNA containing the nucleotide sequence to be copied.
DNA polymerase enzymes
A good supply of all 4 nucleotides.
Primers (synthetic oligonucleotides with sequences that are complementary to the ends of the required piece of DNA).

39. The Method for PCR
The DNA is heated briefly to allow it to unwind and separate.
Then it is cooled to allow primers to bind to the ends of the desired sequence with hydrogen bonds – one primer on each strand of DNA at the 3’ end
DNA polymerase adds new nucleotides from the primer onwards, using the DNA as a template (DNA synthesis).

40. The Method for PCR The DNA has now been doubled.
The process is repeated over and over, doubling the amount of the target DNA each time.
Each cycle takes about 5 mins.