What is DNA sequencing?
Finding the nucleotide sequence for a gene or whole genome.
What is Sanger sequencing?
- Create copies of DNA fragments - extract a DNA sample and heat it to separate the two DNA strands, then cut the strands to create fragments.
- Create complementary DNA fragments - place DNA fragments in a mixture with DNA nucleotides, DNA polymerase, DNA primers and terminating DNA nucleotides. DNA polymerases use DNA primers to attach to the DNA fragments, then create complementary DNA fragments with the DNA nucleotides (that end with terminating DNA nucleotides).
- Analyse complementary DNA fragments - separate fragments by length and since we know the base that each DNA nucleotide ends on, we can work out the original samples DNA sequence.
What are the benefits of High-Throughput Sequencing?
A more recent sequencing technique:
- automated
- very rapid
- much cheaper than the Sanger technique
What are the benefits of DNA sequencing?
- Allows genome-wide comparisons between individuals and between species. This reveals how closely related individuals/species are.
- Allows us to predict the amino acid sequence of genes, and therefore allows us to predict the tertiary structure of the polypeptide that the genes code for.
- Used for synthetic biology - modifying existing DNA sequences to produce specific proteins that can be used as drugs.
What is gel electrophoresis?
A technique used to separate molecules of DNA, RNA or proteins.
- DNA and RNA are separated by mass (length of fragment).
- Proteins can also be separated by mass, determined by the size of their R groups, or the number of amino acids present. Or proteins can be separated by charge, which is determined by the R groups.
What is the process of gel electrophoresis?
- Take a piece of Agar gel and cut a line of holes (wells) into it.
- Submerse the Agar in a buffer solution.
- Load the molecules that they want to separate into one of the wells, and place a negative electrode at the end of the gel near the wells, and a positive electrode at the opposite end.
- Apply an electric current, moving from negative to positive electrode.
- The lighter or more negatively charged a molecule is, the faster it will move across the gel, moving further than heavier/less negatively charged molecules in a given time period.
- Multiple samples can be compared by filling multiple wells in the agar. Different bands are seen by adding a fluorescent dye to the gel, which glows under UV light.
Uses of gel electrophoresis
Used to separate DNA fragments for genome sequencing or DNA profiling.
What is genetic engineering?
- The process of isolating a gene from one organism, and placing it into another organism.
- These organisms can then translate the added gene, because the genetic code is universal.
- It can be used to modify plants, animals and microorganisms.
What is gene therapy?
- When a patients DNA is altered to treat or cure a disease (a use of genetic engineering).
- In somatic gene therapy, a new gene is introduced into somatic body cells, targeting the specific cells in the tissue needs treatment. However as somatic cells eventually die, this treatment has short-lived effects.
- In germ-line gene therapy, the germ is introduced into germ cells (sperm and egg cells). This means that all the cells in the offspring will be altered, therefore the treatment has long-term effects that will be inherited.
Uses of genetic engineering
- Somatic and germ-line gene therapy
- Modify plants eg. soybeans, to give them insect resistance
- Modify pathogens for research into developing new medical treatments
- Used in pharming - an animals DNA is altered so that they produce human proteins for medicine, or they develop human diseases so that new pharmaceuticals can be tested on them.
What are the ethical issues associated with genetic engineering?
- Pharming raises ethical issues (due to modifying animals to act as models for human diseases and putting human genes into animals).
- When companies modify an organism, they will usually patent this modification (the company prevents other companies from replicating their modification for a limited time period), meaning the company can charge a large amount of money for the modified organism, eg. genetically modified seeds, preventing poorer farmers from successfully growing crops.
How are DNA fragments produced using restriction endonucleases?
Taking an organisms DNA and cutting the DNA fragment you want, out of it:
1. There are around 3000 different types of restriction endonuclease, each able to recognise a specific sequence of bases (the recognition sequence).
2. When the restriction endonuclease recognises the recognition sequence, it cuts the DNA at that point.
3. A DNA molecule may have repeats of the same recognition sequence. Therefore the restriction endonuclease cuts the DNA molecule at both locations, producing a fragment of DNA.
4. Restriction endonuclease cut through the DNA molecule by breaking the phosphodiester bonds between adjacent nucleotides.
5. Some restriction endonuclease cut straight through the DNA molecule (so it has straight chain ends), however other restriction endonucleases do not, creating over hanging strands that are complementary to each other.
How are DNA fragments produced using reverse transcriptase?
- Reverse transcriptase is able to reconstruct the DNA sequence that corresponds to the mRNA bases, creating DNA fragments:
1. mRNA is single stranded, so has exposed bases. Reverse transcriptase binds to the mRNA molecule, and add complementary DNA nucleotides to the exposed complementary bases.
2. Reverse transcriptase continues to move along the mRNA molecule, adding DNA nucleotides as it goes, until it has formed a new, complementary DNA stand (complementary DNA/cDNA).
3. Another enzyme destroys the mRNA molecule, leaving us with single-stranded complementary DNA.
4. DNA polymerase then moves along the complementary DNA stand, adding DNA nucleotides with complementary bases and forming a second DNA strand.
Why do genes need to be copied?
- In order to make a lot of a particular protein, you need a DNA fragment that containing the gene that codes for this protein.
- This gene needs to then be copied, so that a lot of the protein can be made.
- Genes are copied using genetic engineering.
How are genes copied?
Genes can be copied inside a living organism:
- the DNA fragment containing the desired gene is transferred to a living organism (host cell - usually a bacterium), genetically modifying it.
- once the DNA fragment has been transferred, the bacterium multiplies, which it can do very quickly, resulting in a much larger amount of bacterium that produce the desired protein.
- this protein is then harvested and is ready for use.
- insulin is produced in this way.
What are vectors?
- Molecules that carry DNA fragments into a host cell - so that the host cell does not think the DNA fragment is foreign and attack it.
- When the host cell is a bacterium, a commonly used vector is a plasmid (since bacterium already contain plasmids).
- This plasmid can then replicate within the plasmid, and can replicate further when the bacterium itself multiplies.
How are DNA fragments inserted into plasmids?
- The same restriction endonuclease that was used to make the DNA fragment is used to cut the plasmid.
- This produces overhanging strands (sticky ends) on the plasmid, that are complementary to the overhanding strands (sticky ends) on the DNA fragment.
- Therefore the DNA fragment and plasmid slot together with hydrogen bonds between the complementary bases.
- DNA ligase then catalyses the formation of phosphodiester bonds between these adjacent nucleotides.
- This creates a recombinant plasmid.
How are recombinant plasmids inserted into bacterium? (two methods)
- Calcium ions and heat shock:
- Calcium ions are added to a mixture of recombinant plasmids and bacterium.
- Calcium ions attract the negatively charged phosphate group in the DNA backbone, and the negatively charged phosphate heads in the cell surface membrane, bringing the plasmid closer to the cell surface membrane.
- The temperature of the mixture is then increased (heat shock), which increases the space between adjacent phospholipids, creating gaps for the plasmid to pass though. - Electroporation:
- Scientists add a small electric current to the bacteria, which increases the number of pores in the membrane.
- This makes it easier for the plasmid to travel through.
The process of adding a plasmid to a host cell is called transformation.
What issues can occur when DNA is added to the plasmid?
- While inserting a DNA fragment into a cut plasmid, it is possible for the cut plasmid to join back together without incorporating the DNA fragment.
- The DNA fragments could join to each other, instead of the plasmid. If this DNA molecule gets into a bacterium, it will be identified as foreign and destroyed.
What issue can occur during transformation?
- Despite using calcium ions and heat shock, or electroporation, the bacterium still may not take up the plasmid.
- This could be due to the size of the plasmid, or the scale of the temperature rise during heat shock.
How do you identify the bacteria that contains the recombinant plasmid?
- Beginning genetic engineering using a plasmid that contains a gene for antibiotic resistance (a marker gene):
- when a DNA fragment is added, it disrupts the gene, stopping it from functioning
- therefore any plasmid that has taken up the DNA fragment won’t provide resistance to the antibiotic
- when the bacteria are placed in a plate containing the antibiotic, only the bacteria containing the recombinant plasmid will die.
What is the risk of using an antibiotic resistance marker gene to identify the bacteria that contains the recombinant plasmid?
- It is possible that these marker genes could be transferred to other, disease causing bacteria.
- Meaning that if we are infected with this bacteria, we cannot get rid of them with the antibiotic.
- Therefore different marker genes have been being used.
What other marker genes can identify the bacteria that contains the recombinant plasmid?
- Fluorescent marker gene:
- once taken into the bacterium it makes the bacterium glow under UV light
- the addition of a DNA fragment disturbs this gene, meaning that bacteria containing the recombinant plasmid won’t glow under UV light. - Enzyme marker gene:
- this gene produces an enzyme that causes an observable effect eg. lactase gene (turns colourless substrates blue)
- the addition of the DNA fragment disturbs this gene, so the enzyme cannot be produced, therefore the bacteria that do not have the observable effects are the bacteria that contain the recombinant plasmid.
Once the bacteria with the recombinant plasmid has been identified, they are collected, multiplied and left to produce a lot of the protein we want.
What is the Polymerase Chain Reaction (PCR)?
- Takes place in a machine
- Produces copies of DNA fragments in a continuous cycle
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