IBDP Biology - Genomes and gene technologies

In this IBDP Biology topic, you will learn genomes and gene technologies.

Genome can mean all genes of an individual organism or all genes in a population of organisms.

Gene technologies used to study genes and their functions include:

• Polymerase chain reaction (PCR)
• Cutting out DNA fragments using restriction enzymes
• Gel electrophoresis
• DNA probes

1. If a whole genome needs sequencing: use PCR to make multiple copies of all the DNA. Squeeze through a tiny hole under high pressure. Cuts DNA into 2000- 10000 base pairs. These can be shortened to make it easier to sequence them.
2. Make multiple labelled copies of these lengths of DNA. They are mixed with a primer (up to 20 base pairs of DNA, complimentary to the start of the DNA fragment you want), free nucleotides, DNA polymerase (to create new DNA strands) and nucleotides labelled with fluorescent dye. Each base has it’s own colour and each labelled nucleotide with stop the chain from growing.
3. The DNA polymerase attaches free nucleotides to the lengths of DNA. Most of the copies wont be complete due to the labelled nucleotides. This means lots of different length chains.
4. This is then separated using electrophoresis so the DNA is drawn towards the positive end of a capillary tube.
5. A computer records the colours as they pass, the shorter the chain, the faster it travels, so if there are enough fragments, the computer will be able to work out the sequence of bases in that particular length of DNA.
6. This is then done for every piece of DNA and then a computer program works out the overall sequence of nucleotides in the whole genome.

PCR can be used to make millions of copies of a DNA fragment in just a few hours.

1. DNA is denatured by heating to 95˚C, to break hydrogen bonds of the double helix of DNA, so the base pairs are exposed.
2. The mixture is then cooled to between 50˚C - 65˚C so the primer (up to 20 pieces of DNA complimentary to the start of the DNA fragment you want) can bind (anneal) to the strands.
3. The mixture is then heated to 72˚C so DNA polymerase can DNA strand.

4. The DNA polymerase lines free nucleotides alongside each template strand, base pairing means new complementary strands are formed.

5. Two new copies of each DNA strand are formed from one cycle of PCR.

6. The cycle starts again with the mixture being heated to 95˚C and all four strands are used as templates.

7. Each PCR cycle doubles the amount of DNA from the previous cycle.

This separates different DNA fragments according to their lengths.

• A tank containing pure agar (agarose gel) is set up.
• A direct current is passed through the gel and the DNA fragments (which carry a small negative charge) are attracted to the anode (positive electrode)
• The smaller the fragment, the faster they move.
• Labelled nucleotides allow us to use time to sort longer DNA strands from shorter ones.

You can get a DNA fragment from a length of DNA using restriction enzymes.

1. Cut DNA backbones
2. Restriction nucleoases that cut DNA at specific points.
3. Each particular enzyme cuts DNA at a different point- (the restriction site)
4. Less than 10 bases long.
5. Catalyse a hydrolysis reaction that breaks the phosphate – sugar backbone of the double helix. Leaves exposed bases called sticky ends.

• Catalyses condensation reaction that joins the phosphate- sugar backbones in the helix together.
• Both pieces of DNA need to have been cut with the same restriction enzyme, so the sticky ends are complimentary and can hydrogen bond together. DNA ligase then seals the backbone.
• The resulting DNA is recombinant DNA.

• Genetically engineered to produce beta carotene which is used to produce vitamin A. Being used to reduce vitamin A deficiency in places like south Asia and parts of Africa

1. Psy gene is extracted from daffodil and crtl gene extracted from soil bacterium using restriction enzymes
2. A plasmid is removed from Agrobacterium tumefaciens and cut open using the same restriction enzymes
3. The genes and a marker gene are inserted into the plasmid
4. The recombinant plasmid is put back into the bacterium
5. Rice plants are incubated with the transformed bacteria which infect the plant cells
6. Rice plants are grown on a selective medium so only the transformed plants will be able to grow because they contain the marker needed to grow on this medium.

Determines order of bases in a section of DNA

1. Single stranded DNA, primer, DNA polymerase, free nucleotides and fluorescently labelled nucleotides are mixed together and put in four tubes. A different modified nucleotide is added to each of the tubes.
2. They undergo PCR, which produces many strands of DNA. They’re different lengths because they terminate at different points depending on where the modified nucleotide was added
3. The DNA fragments are separated by electrophoresis and visualised under fluorescent light
4. The complimentary base sequence can be read from the gel. The smallest nucleotide is at the bottom and by reading the bands from top to bottom, you can build the DNA sequence one base at a time

This is the end of the topic

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Drafted by Eva (Biology)

Photo references:

1. https://en.wikipedia.org/wiki/Polymerase_chain_reaction
2. https://www.youtube.com/watch?v=YF0UaIB0QpE
3. https://www.addgene.org/protocols/dna-ligation/
4. https://www.npr.org/sections/thesalt/2013/03/07/173611461/in-a-grain-of-golden-rice-a-world-of-controversy-over-gmo-foods