1
Q
What is the genome of an organism?
A
All the genetic material it contains
2
Q
What are exons?
A
The genes of out DNA that code for proteins
3
Q
What are introns?
A
The large non-coding regions of DNA that are removed from mRNA before it is translated into a polypeptide chain
4
Q
What is satellite DNA?
A
- Short sequences of DNA that are repeated many times
- The repeats always appear in the same positions on chromosomes but the number of repeats of each mini/micro satellite varies between individuals. The more closely related you are to someone, the more likely you will have similar latterns
5
Q
What is a mini satellite piece of DNA?
A
- A sequence of 20-50 base pairs which is repeated
- Aka variable number tandem repeats
6
Q
What is a microsatellite piece of DNA?
A
- A region of 2-4 bases repeated
- Aka short tandem repeats
7
Q
What are the five main stages of producing a DNA profile?
A
- Extracting the DNA
- Digesting the sample
- Separating the DNA fragments
- Hybridisation
- Seeing the evidence
8
Q
What does extracting the DNA for a DNA profile involve?
A
- Extracting DNA from a tissue sample and using polymerase chain reaction (PCR) to amplify a tiny sample into a sample big enough to develop a profile
9
Q
What does digesting the sample for a DNA profile involve?
A
- The strands of DNA are cut into small fragments using special enzymes called restriction endonucleases
10
Q
How do restriction endonucleases work?
A
- Different restriction endonucleases cut DNA at a specific nucleotide sequence, known as a recognition site
- Each restriction endonuclease has an active site complementary to a specific, short nucleotide sequence, known as a recognition site, where they make the cuts
11
Q
What does separating the DNA fragments for a DNA profile involve?
A
- The cut fragments of DNA need to be separated to form a clear pattern, which is done using electrophoresis
- Electrophoresis utilises the way charged particles move through a gel medium under the influence of an electric current. The gel is then immersed in alkali to seepage the DNA double strands into single strands. The single stranded DNA fragments are then transferred onto a membrane by Southern blotting
12
Q
What does hybridisation for a DNA profile involve?
A
- Radioactive/fluorescent DNA probes are added in excess to the DNA fragments on the membrane
- The DNA probes are short DNA or RNA sequences complementary to a known DNA sequence. They hind to the complementary strands of DNA under particular conditions of pH and temperature. This is called hybridisation
- The excess probes are washed off
13
Q
What does seeing the evidence of a DNA profile involve?
A
- If radioactive labels were added to the DNA probes, X ray images are taken on the paper/membrane
- If fluorescent labels were added to the DNA probes, the paper/membrane is placed under UV light so the fluorescent tags flow. The fragments give a pattern of bars - the DNA profile - which is unique to every individual expect identical twins
14
Q
What is PCR full name?
A
Polymerase chain reaction
15
Q
What is PCR?
A
- A process by which DNA is amplified (replicated), allowing scientists to produce a lot of DNA from the tiniest original sample
16
Q
What is placed in a PCR machine for PCR to take place?
A
The DNA sample that is going to be amplified, an excess of the four nucleotide bases, primer DNA sequences, and the enzyme DNA polymerase. This is mixed in a vial which is placed in a PCR machine (aka a thermal cycler)
17
Q
What is a PCR machine also known as?
A
A thermal cycler
18
Q
What is the first stage of PCR? (First heating)
A
- The DNA mixture is heated to 95 degrees for 30 seconds, to break the hydrogen bonds between the two strands of DNA which denatures the DNA, and the strands seperate.
- DNA polymerase doesn’t denature even at high temperatures- important as it can be reused in many cycles.
19
Q
What is the second stage of PCR? (Cooling)
A
- The mixture is then cooled to between 55 and 65 degrees so that the primers can bind (anneal) to end of the DNA the strands
- They are needed for the replication of the strands to occur
20
Q
What is the third stage of PCR? (Second heating)
A
- The reaction is heated to 72 degrees for at least 1 minute so DNA polymerase can work best at its optimum temperature
- Taq polymerase is used
- The DNA polymerase lines up free DNA nucleotides alongside each template strand. Complementary base pairing means new complementary strands are formed. It adds bases to the primer
- Two new copies of the fragment are formed, the cycle starts again, using double the fragments as the first cycle.
21
Q
Why is taq polymerase used in the third stage of PCR?
A
The most commonly used polymerase is Taq polymerase as it comes from a thermophilic bacterium Thermus aquaticus which means it does not denature at the high temperature involved during the first stage of the PCR reaction and secondly, its optimum temperature is high enough to prevent annealing of the DNA strands that have not been copied yet
- Allows higher rate of DNA replication
- Optimum is 72 degreees
- Withstands higher temps than normal DNA polymerase
22
Q
How is electrophoresis carried out on proteins?
A
The same process but proteins can be positively or negatively charged so, before they undergo electrophoresis, they’re mixed with a chemical that denatures the proteins so they’ll have the same charge. This has many uses like identifying proteins in bodily liquids, to help diagnose disease.
23
Q
How is electrophoresis carried out?
A
- Put the lid on the gel box and connect the gel box leads to a power supply, and set it to the correct voltage, which causes an electrical current to be passed through the gel
- DNA fragments are negatively charged so they’ll move through the gel towards the positive electrode at the far end of the gel (anode). Small DNA fragments move faster and travel further through the gel, so DNA fragments separate according to size.
- Let the gel run for about 30 minutes, or until the dye is about 2cm from the end of the gel.
- Remove the gel tray and tip off any excess buffer solution
- Wearing gloves, stain the DNA fragments by covering the surface of the gel with a staining solution then rinsing the gel with water, the bands of the different DNA fragments will now be visible. The bands are measured in bases.
24
Q
What is the process of setting up electrophoresis?
A
- Commonly performed using agarose gel that has been poured into a gel tray and left to solidify. A row of wells is created at one end of the fell.
- The gel tray is put into a gel box, making sure the wells are closet to the negative electrode on the gel box.
- Add buffer solution to the reservoirs at the sides of the gel box so the surface becomes covered in buffer solution
- Using a micropipette, add loading dye to each fragmented DNA sample which helps them sink to the bottom of the wells and makes them easier to see
- Add a set volume of DNA samples to the wells, ensuring your micropipette is in the buffer solution on top so you don’t pierce the well.
- Record which DNA samples to you have added to each well
25
What is electrophoresis used for?
It uses an electrical current to separate out DNA fragments, RNA fragments or proteins depending on their size
26
How is DNA profiling used in forensic science?
- PCR and DNA profiling is performed on the traces of DNA left at the crime scheme which can be compared to that of a sample taken from a suspect, or can be identified from a criminal database
27
How is DNA profiling used to identify individuals at risk of developing particular disease?
- Certain non-coding micro satellites or the repeating patterns they make, have been found to be associated with an increased risk of particular diseases
- These genes can be identified and observed in DNA profiles
28
How else can DNA profiles be used?
- Proving paternity of a child
- Identifying species to which an organism belongs
- Evolutionary relationships between different species
29
What is Sanger sequencing?
- One of the first methods used to sequence DNA/the genome of an organism
- The chain termination method of DNA sequencing which uses modified nucleotides called terminator bases
30
What is a terminator base?
- Used in Sanger DNA sequencing
- Modified versions of the four nucleotide bases, adenine, thymine, guanine and cytosine, which will stop DN synthesis when they are included
- An A terminator base will stop DNA synthesis at the location that an A base would be added, etc.
- The terminator bases are also given coloured fluorescent tags - A is green, G is yellow, T is red and C is blue
31
How is Sanger sequencing carried out? (The capillary method)
1. The DNA for sequencing is mixed with a primer, DNA polymerase, an excess of normal nucleotides and terminator bases
2. The mixture is placed in a thermal cylinder - and PCR takes place
3. During PCR, each time a terminator base is incorporated instead of a normal nucleotide (at 60 degrees when DNA polymerase is building the new DNA strand), the synthesis of DNA is terminated as no more bases can be added
4. The terminator bases are present in lower amounts and are added at random, and this results in many DNA fragments of different lengths depending on where the chain terminating bases have been added during the process. After many cycles all of the possible DNA chains will be produced with the reaction stopped at every base.
5. The DNA fragments are separated according to their length by capillary sequencing, which is like gel electrophoresis in minute capillary tubes. The fluorescent markers on the terminator bases are used to identify the final base on each fragment. Lasers detect the different colours and thus the order of the sequence
6. The order of bases in the capillary tubes shows the sequence of the new, complementary strand of DNA which has been made, which is used to build up the sequence of the original DNA strand.
32
What is high-throughput sequencing?
- A newer and much faster method of DNA sequencing rather than Sanger sequencing
- Advances in technology have enabled the development of high-throughput sequencing methods which allow scientists to rapidly sequence the genomes of organisms
- It allows hundreds of sequences to be run in parallel, is automated and faster. The tube contains all the terminator bases, each with a different fluorescent label, and a machine reads the sequence to you giving a computer read pit
33
What is genetic engineering?
The manipulation of an organism’s DNA
34
What are transformed organisms?
Organisms that have had their DNA altered by genetic engineering
35
What is recombinant DNA?
DNA formed by joining together DNA from different sources - this is what genetically modified organisms have.
36
What does genetic engineering involve?
Extracting a gene from one organism and inserting it into another organism. Genes can also be manufactured instead of extracted. The organism with the inserted gene will then produce the protein coded for by that gene.
37
What is a transgenic organism?
An organism that had been genetically engineered to include a gene from a different species.
38
What is the first stage of genetic engineering?
The DNA fragment containing the desired gene is obtained, the fragment is isolated using restriction enzymes.
39
What is the second stage of genetic engineering?
The DNA fragment (with the gene in) is inserted into a vector, which is used to transfer DNA into a cell.
40
What vectors can be used in genetic engineering?
Plasmids or bacteriophages
41
How is the DNA fragment inserted into a vector?
- The vector DNA is cut open using the same restriction enzyme that was used to isolate the DNA fragment containing the desired gene.
- This means the sticky ends of the vector are complementary to the sticky ends of the DNA fragment.
- The vector DNA and DNA fragment are mixed together with DNA ligase. DNA ligase joins up the sugar-phosphate backbones of the two. This is ligation.
- The new combination of the bases in the DNA (vector DNA + DNA fragment) is recombinant DNA
42
What is the third stage of genetic engineering?
The vector transferring the gene into the bacteria
43
How are plasmid vectors taken in by bacterial cells using electroporation?
- A suspension of the bacterial cells is mixed with the plasmid vector and placed in a machine called an electroporator.
- The machine is switched on and an electrical field is created in the mixture, which increases the permeability of the bacterial cells is membranes and allows them to take in the plasmids.
44
How are bacteriophage vectors taken up by bacterial cells?
- The bacteriophage will infect the bacterium by injecting its DNA into it. The phage DNA (with the desired gene) then integrates into the bacterial DNA.
45
How is insect-resistance created in plants?
Using genetic manipulation to insert a gene into the cells of the plant which makes them resistant to insect plants,
46
Why were soybeans genetically modified to be resistant to some insects?
Soybeans are an important food source across the world, but yields can be greatly reduced by insect pests that feed on the plant. The gene codes for a protein that is toxic to some of the insects that feed on the plant.
47
How are soybeans GM?
- Using restriction enzymes to insert a desire gene from a bacteria into a plasmid. The plasmid is put back into the bacteria. The plant cells are deliberately infected with the transformed bacteria. The desired gene gets inserted into the soybean plant cells DNA.
48
What is a positive of GM plants?
Reduces the amounts of chemical pesticides that farmers use on crops, which can harm the environment.
49
What are negative ethical issues of GM plants?
It may encourage monoculture (where only one type of crop is planted). Monoculture decreases biodiversity and could leave the whole crop vulnerable to disease as all the plants are genetically identical.
50
What is pharming?
When medicinal drugs can be produced using genetically modified organisms, such as animals.
51
How can be pharming carried out?
- DNA fragments that code for production of specific proteins are injected into the animal embryo
- The embryo is implanted into a female, and when the offspring is born it is tested to see if it produces the protein, e.g, in its milk
- If it does produce the protein, selective breeding is used to produce a herd of the animal that produce the protein in their milk
- The protein can be extracted from the milk to produce a drug
52
What are positive ethical issues of pharming?
Drugs can be made in large quantities compared to other methods of production, making them more avaliable to people
53
What are negative ethical issues of pharming?
- Manipukating an animal’s genes could cause harmful side effects for the animal, and using an animal in this way enforces that animals are assets that can be treated however we choose, animals can’t give consent
54
How can genetic engineering be carried out on pathogens to find treatments to disease?
- Bacteria can be genetically modified to produce useful substances to people, like insulin and vaccines
55
What ethical issues does genetic engineering of pathogens raise?
- It has helped to cure diseases, as previously untreatable disease can now be treated, reducing suffering
- However:
- There are worries the scientist researching the pathogen could become infected and cause a mass outbreak of disease
- The genetically modified pathogen could revert back to its original form and cause an outbreak of disease
- In the wrong hands, knowledge of how to genetically engineer dangerous pathogens could be used maliciously to create agents of biowarfare
56
What is patening and technology transfer for genetically modified products?
- Scientists working for different institutions often share their knowledge and skills in the field, so that globally, beneficial genetically modified products can be created at a faster rate. This is technology transfer
- A group of scientists may want to gain legal protection for their genetically modified products, by getting a patent which means by law they can control who uses the product and how.
57
What are the positives of patening?
- This has positives like the owner of the patent getting money from selling the product to develop more methods. It also encourages scientists to compete to be the first to come up with new beneficial genetic engineering ideas
58
What are the negatives of patenting?
There are negatives like farmers in poorer countries not being able to afford patented genetically modified seeds. Some patents also mean they are not legally allowed to plant and grow any of the seeds from the crop without paying again. Some patents mean they can’t plant and grow seeds from the crop without paying again. This is unfair and the big companies that own the patents are benefitting the most without regard to help farmers in poorer countries.
59
What is gene therapy?
Altering alleles to cure genetic disorders
60
How can a genetic disorder caused by two recessive alleles be fixed?
Adding a dominant allele to make up for them
61
How can a genetic disorder caused by a dominant allele be fixed?
- ‘Silence’ the dominant allele e.g. putting DNA in the middle so it doesn’t work anymore
62
How is the new allele inside the cell in gene therapy?
Using vectors, like alternated viruses, plasmids or liposomes
63
What are the two types of gene therapy?
- Somatic therapy
- Germ line therapy
64
What is somatic gene therapy?
- Altering the alleles in body cells, particularly the cells that are most affected by the disorder
- Replacing the mutant allele with a healthy allele in affected somatic (body) cells
65
What are the drawbacks of somatic gene therapy?
- It is only a temporary treatment as somatic cells have a limited life and are replaced by stem cells, which will have the faulty allele
- A treated individual will also still pass the faulty allele onto any children they have
66
What is germ line therapy?
- Altering the alleles in sex cells
- Inserting a healthy allele into the germ cells, or into an embryo immediately after fertilisation
- The individual would be born healthy with the normal allele in place and would pass it into their own offspring
67
What are the positives of germ line cell therapy?
- The individual would be born healthy with the normal allele in place
- The individual would pass the healthy allele onto their own offspring
68
What are positives of gene therapy?
- Prolongs lives of people with genetic disorders
- Gives them a better quality of life
- Carriers might be able to conceive a baby without the disorder with germ line therapy
- Germ line could decrease the number of people that suffer from genetic disorders
69
What are negative ethical issues of gene therapy?
- The treatment could be used in other ways than medical treatment like choosing desirable or cosmetic characteristics in their offspring
- Risk of harm in the patient e.g. overexpression of genes
- Expensive and short lived (somatic)
- Difficult to get into specific body cells
- Body could identify vectors as foreign bodies and start an immune response against them
70
How has sequencing genes led to synthetic biology by showing polypeptide structure?
- Animo acids are coded for by triplets of bases in a gene.
- This means that by sequencing a gene, the sequence of amino acids that a gene codes for and so the primary structure of a polypeptide can be predicted.
- This has allowed us to create biological molecules from scratch and so has led to the development of an area of biology called 'synthetic biology.
- Synthetic biology is a large field that includes:
building biological systems from artificially made molecules (e.g. proteins) to see whether they work in the way we think they do, redesigning biological systems to perform better and include new molecules, designing new biological systems and molecules that don't exist in the natural world, but could be useful to humans, eg. energy products (fuels) and drug products.
71
How can sequences genes and genomes be compared?
- Gene sequences and whole genome sequences can be compared between organisms of different species and betveen organisms of the same species.
- This is a complicated process which is made easier with the use of computers - involves computational biology (using computers to study biology) and bioinformatics (developing and using computer software that can analyse, organise and store biological data).
- There are many reasons why biological research can involve comparison of gene sequences and genomes
72
How has bioinformatics and computational biology contributed to research in genotype-phenotype relationships?
- It's useful to be able to predict an organism's phenotype by analysing its genotype.
- Bioinformatics has allowed the scientists to compare all the data and identify genotype-phenotype correlations — this could help in the treatment of syndromes by using gene sequencing to predict what health problems the person is likely to face.
73
How has bioinformatics and computational biology contributed to research in epidemiological studies?
- Epidemiology is the study of health and disease within a population — it considers the distribution of a disease, its causes and its effects.
- Some gene mutations have been linked to a greater risk of disease.
- Computerised comparisons between the genomes of people that have a disease and those that don't can be used to detect particular mutations that could be responsible for the increased risk of disease.
74
How has bioinformatics and computational biology contributed to research in evolutionary relationships?
- All organisms evolved from shared common ancestors (relatives). Closely related species evolved away from each other more recently and so share more DNA.
- Whole genomes of different species can be sequenced and then analysed using computer software to tell us how closely related different species are. E.g. the genomes of humans and chimpanzees are about 94% similar.
- Comparing the genomes of members of the same species can also tell us about evolutionary relationships. - For example, when different groups of early humans separated and moved to different parts of the world, their genomes changed in slightly different ways. By using computers to compare the genomes of people from different parts of the world, it's possible to build up a picture of early human migration.
