Fragments of DNA can be produced by • conversion of mRNA to cDNA, using reverse transcriptase • cutting DNA at specific, palindromic recognition sequences using restriction endonucleases • the polymerse chain reaction (PCR). Fragments of DNA produced by any of the above methods can be used to clone genes by in vivo and in vitro techniques. In vivo cloning. The use of restriction endonucleases and ligases to insert a gene into vectors, which are then transferred into host cells. The identification and growth of transformed host cells to clone the desired DNA fragments. The importance of “sticky ends”. In vitro cloning. The use of the polymerase chain reaction (PCR) to clone directly. The relative advantages of in vivo and in vitro cloning. The use of recombinant DNA technology to produce transformed organisms that benefit humans. Candidates should be able to • interpret information relating to the use of recombinant DNA technology • evaluate the ethical, moral and social issues associated with the use of recombinant technology in agriculture, in industry and in medicine • balance the humanitarian aspects of recombinant DNA technology with the opposition from environmentalists and anti-globalisation activists.

Genes are often cloned in order to produce proteins, these can be used to treat diseases. This is better than taking already made proteins because:

• It is cheaper
• There is no risk of rejection by the immune system
• There is no risk of infection

DNA fragment production- isolation
Fragments of DNA can be used to clone genes. Fragments can be produced in several ways:

• Reverse transcriptase: this is an enzyme which can make DNA from RNA. mRNA is extracted and reverse transcriptase binds complementary bases to make a strand of complementary DNA (cDNA). DNA polymerase adds complementary base pairs to create the other strand of DNA. Reverse transcriptase is taken from retroviruses like HIV.

• Restriction endonucleases: these are enzymes that cut DNA. They cut it at a sequence of bases specific to the particular restriction endonuclease known as the recognition sequence. They are taken from bacteria which have them to inhibit viruses. They either cut it straight leaving a blunt end or cut along in between bases leaving a sticky end.

In vivo cloning
Bacteria have some DNA in small circular strands called plasmids. If a gene is added to a plasmid, the bacteria will produce the protein that that gene codes for. So you could add the gene for insulin to a plasmid and the bacteria will make insulin.

This is done by cutting DNA from a human cell with a certain restriction endonuclease. The same one is used to cut the plasmid in the gene for resistance to the antibiotic tetracycline (for example). This will mean that the plasmid and DNA have complimentary sticky ends and so can be joined together by DNA ligase. This is called insertion, it does not always work. When the two are joined together they are known as recombinant DNA.

The plasmid is the reinserted into the bacteria by using calcium ions and temperature changes to make the cell membrane permeable. This is called transformation, it does not always work.

To test if plasmids have been taken up (identification) scientists use plasmids that have the gene for resistance to the antibiotic ampicillin on, they then put the bacteria on ampicillin, those that die have not taken up the plasmid because they are not resistant, those the survive have the plasmid as they are resistant.

To test if the plasmids successfully took up the DNA in the first place gene markers are used:

• Antibiotic resistance: after the test for ampicillin, the bacteria that are left alive are replicated (replica plating) and grown on tetracycline, those that die have plasmids which have taken up the new DNA because it is in the middle of the gene for resistance to tetracycline so it doesn't work.
• Fluorescence: plasmids are cut where the gene for fluorescence is and the DNA inserted. This means for plasmids that have taken up the gene it will have stopped the gene for fluorescence working and the bacteria will not fluoresce.
• Enzymes: plasmids are cut where the gene for lactase is and the DNA inserted. This means for plasmids that have taken up the gene it will have stopped the gene for lactase working and so bacteria will test negative for its presence (indicator will remain colourless instead of turning blue).

In vitro cloning
Cloning outside of organisms is done by polymerase chain reaction (PCR):

• DNA is heated to 95°, by a thermocycler, which makes the two strands separate
• Primers (which are short sequences of DNA that are complimentary to the ends of the DNA) join the ends of DNA when it is cooled to 55°
• DNA polymerase (which can not make a DNA without a start point provided by primers) can then add free complimentary nucleotides to the DNA at its optimum temperature of 72°
• Now each single strand of DNA has become a double strand
• The process can be repeated, doubling the amount of DNA each time

Recombinant DNA
Organisms that have DNA from other organisms in are called transgenic or GM (genetically modified). They have many uses:

• Improving yield: resistance to disease; toxins that dispel pests; tolerance to environmental conditions (salt, temperature); tolerance to herbicides
• Producing medicines (plant pharming), hormones, vaccines, antibodies, antigens and antibiotics
• Increasing the nutritional value of food
• Manufacturing enzymes
• Making meat more economically viable: faster and larger growing animals; resistance to disease
• Bacteria that break up harmful substances

Transgenic animals are made by taking an egg from an animal and fertilising it with a sperm and then adding DNA. The embryo is then implanted into a womb. When the animal grows up it is bred with other animals with the same modifications to make a population that all have the same modification. They may be modified to improve the animal, by for example increasing resistance, or they may be modified to produce a protein for example to make antibodies in their milk or eggs.

One way in which adding DNA is used, is to prevent organisms from producing a protein, this is done, for example, in tomatoes. DNA is added which has the opposite base sequence to the DNA for a protein that isn't wanted, this means the mRNA it makes is complimentary to the mRNA of the protein that isn't wanted and so will bind with it, meaning it can't be translated to create the protein.

There are negatives to using recombinant DNA:

• Properties may be passed to organisms where it is harmful (e.g. herbicide resistance in a weed or antibiotic resistance in pathogens)
• Organisms may affect the environment (biodiversity)
• Modifications will have effects on the metabolic pathways of cells
• GM DNA may be harmful when it mutates
• Could reduce variety in populations (which would reduce evolution)
• Could lead to eugenics (selection of races)
• There could be economic consequences of crops being able to grow in different countries
• Some people see it as immoral to mess with nature
• It is expensive