3.5 Genetic Modification And Biotechnology

Techniques for gene transfer to bacteria (using restriction endonucleases and DNA ligase)

- A copy gene being transferred is required - Easier to obtain mRNA transcripts of genes than the gene itself (reverse transcriptase)

Why is Taq DNA Polymerase used?

- Because it can resist the brief period at 95 degrees Celsius used to separate the strands - Optimum temperature is 72 degrees celsius - At this temperature it adds about 1000 nucleotides per minute

DNA ligase

- Enzyme that joins DNA molecules together by making sugar-phosphate bonds between the molecules - Can be used to seal nicks in each sugar-phosphate backbone of DNA

Reverse transcriptase

- Enzyme that makes DNA copies of RNA molecules called cDNA. - It is used to make the DNA needed for gene transfer from mRNA

When is the PCR used?

- Forensic investigations - DNA extracted from fossils - Paternity investigations

Bt Corn Monarch Butterflies

- Gene coding for Bt toxin transferred from the bacterium Baciullus thuringiensis in maize - Larvae of monarch butterflies feed on milkweed - These grow close to maize (maize pollen goes on it) - Butterflies might be poisoned

GM crops

- Genes from snapdragons (flower) transferred to tomatoes to produce purple fruits - Golden rice: transfer of three genes (2 from daffodil plants and 1 from a bacterium) - yellow pigment beta carotene is produced in the rice grains.

Clones

- Groups of genetically modified organisms derived from a single original parent cell. - Producing genetically identical organisms - Human zygote divides into two cells---> develop into separate embryos---> different individuals obtained (twins)

Where are samples placed?

- In wells cast in a gel - Gel is immersed in conducting fluid and an electrical field is applied - Molecules with negative and positive charges move in opposite directions. - Proteins may be positively and negatively charged.

What is the gel made of?

- Mesh of filaments that resists the movement of molecules in a sample

Restriction Enzymes

- Or Endonucleases - Cut DNA molecules at specific base sequence - Cut open plasmids + desired genes from larger DNA molecules - Some can cut the two strands of DNA at different points - This leaves sticky-ends - These have complementary base sequences so can be linked together pieces of DNA (H bonds between bases)

Risks of Genetic Modification

- Paul Berg planned an experiment: DNA from the monkey virus SV40 inserted into the bacterium E. Coli. - SV40: causes cancer in mice - E. Coli: present in human intestine. - Concerns were expressed that it might cause cancer in humans. - Bans for GMO's in some countries

Benefits of GM crops

- Pest-resistant (genes for making toxins transferred in the plants) Less insecticide - Reduces the need for plowing and spraying crops (less fuels needed) - Shelf-life of fruits and vegetables can be improved - Nutritional value of crops can be improved (e.g. increasing vitamin content) - Varieties of crops could be produced lacking allergens or toxins that are naturally present in them. - Can be edible vaccines (in the future) - Resistant to draught, cold and salinity, increases total yield. - Gene for herbicide resistance. - Crop varieties can be produced that are resistant to diseases

PCR

- Polymerase Chain Reaction - Used to make large numbers of copies of a selected DNA sequence - Only small fragment of DNA is needed at the start - Doubles amount of DNA (one cycle)

Risks of GM crops

- Proteins produced by transcription or translation could be toxic or cause allergic reactions - Antibiotic resistance genes used as markers during gene transfer could spread to pathogenic to pathogenic bacteria. - Transferred genes could mutate and cause unexpected problems that were not risk-asessed during development of GM crops. - Non-target organisms could be affected by toxins that are intended to control pests in GM crop plants - Genes transferred to crop plants to make them herbicide resistant could spread to wild plants (uncontrollable weeds) - Biodiversity reduced. - Seed is split and germinates to become unwanted: plants must be controlled- this becomes difficult if it contains herbicide genes - Toxin kills insect pests: secondary pests might become resistant to pests. - Farmers cannot save and re-sow GM crops

Plasmid

- Small extra circle of DNA - Over 1,000 base pairs - Bacteria use them to exchange genes (useful in genetic engineering)

Universal genetic code

- When genes are transferred between species, the amino acid sequence translated from them is unchanged. (Same polypeptide is produced). - e.g. Transferring gene to make human insulin to bacterium (large quantities of it can treat diabetes)

DNA molecules for eukaryotes...

... Are too long to move through the gel ... They must be broken up into smaller fragments All DNA molecules are negatively charged, they all move in the same direction

Molecules are separated according to ...

... Their size and charge

PCR steps

1. *DNA* strands *separated* by heating them at 95 degrees celsius for 15 seconds. (H bonds break) 2. *DNA cooled* quickly at 54 degrees celsius. Allows the parent strand to re-anneal to form double stranded DNA 3. A large excess of DNA *primers* is present; they bind rapidly to target sequences. Large excess of primers is present: they prevent re-annealing of parent strand. Copying the single parent strand starts from the primers 4. *Synthesis of double-stranded DNA*: with enzyme Taq DNA Polymerase single strands with primers as template. 5. A cycle can be completed in less than 2 minutes

DNA profiling stages

1. A sample of DNA is obtained 2. DNA sequences are selected and copied by PCR 3. The copied DNA s split into fragments using restriction endonucleases 4. The fragments are separated using gel electrophoresis 5. Produces a pattern of bands that is always the same with DNA taken from one individual *DNA profile* 6. The profiles of different individuals are compared to see which bands are the same and which are different.

Gel Electrophoresis

Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores. The molecules travel through the pores in the gel at a speed that is inversely related to their lengths. This means that a small DNA molecule will travel a greater distance through the gel than will a larger DNA molecule.

Sticky Ends

Single stranded sections

Genetic modification

The transfer of genes from one species to another