1
Q
What are the 5 main stages of DNA profiling?
A
- extracting the DNA
- Digesting the sample
- separating the DNA sample
- Hybridisation
- Viewing results.
2
Q
Explain the first step of DNA profiling, Extracting the DNA.
A
DNA must be extracted from a tissue sample. When DNA profiling was first discovered, a relatively large sample was needed- about 1ug of DNA, approx. 10,000 human cells.
- Now, by using PCR, the tiniest fragment of tissue can give the scientists enough DNA to develop a profile.
3
Q
Explain step 2 of DNA profiling- digesting the sample.
A
- The strands of DNA are cut into small fragments using enzymes called restriction endonucleases.
- Different restriction endonucleases cut DNA at a specific nucleotide sequence known as a restriction/ recognition site.
- All restriction endonucleases make 2 cuts, once through each strand of the DNA double helix.
- Restriction endonucleases have given scientists the ability to cut the DNA strands at defined points in the introns. They use a mixture of restriction enzymes that leave the repeating units or satellites in tact, so the fragments at the end of the process include a mixture of intact mini and micro satellite regions.
4
Q
Explain the third step in DNA profiling- Separating DNA fragments.
A
- To produce a DNA profile, the cut fragments of DNA need to be separated to form a clear and recognisable pattern. This is done through electrophoresis, a technique that 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 separate the DNA double strands into single strands.
- The single stranded DNA fragments are then transferred onto a membrane by southern blotting.
5
Q
Explain the 4th step in DNA profiling- Hybridisation.
A
- Radioactive or fluorescent DNA probes are now added in excess to the DNA fragments on the membrane. DNA probes are short DNA or RNA sequences complementary to a known DNA sequence. They bind to the complementary stands of DNA under particular conditions of pH and temp.
- DNA probes identify the microsatellite regions that are more varied than the larger minisatellite regions.
- Excess probes are washed off.
6
Q
Explain stage 5 of DNA profiling- Viewing the evidence.
A
- If radioactive labels were added to the DNA probes, X-ray images are taken of the paper/ membrane. If fluorescent labels were added to the DNA probes, the paper/ membrane is placed under UV light so the fluorescent tags glow. This is the method most commonly used today.
- Fragments give a pattern of bars- the DNA profile- which is unique to every individual except identical siblings.
7
Q
Explain the process of separation of nucleic acid fragments by electrophoresis.
A
- DNA fragments are put into well in agarose gel strips which also contain a buffering solution to maintain a constant pH.
- In one or more wells, DNA fragments of known lengths are used to provide a reference for fragment sizing.
- When an electrical current is passes through the electrophoresis plate, the DNA fragments in the wells at the cathode end move towards the positive anode at the other end. This is due to the negatively charges PO4^3- groups in the DNA fragments.
- The rate of movement depends on the length of the DNA fragments.- the gel has a mesh-like structure that resists the movement of molecules.
- Smaller fragments can move faster, therefore over a certain time period, smaller fragments will move further.
- When the faster smallest fragments reach the anode, the electrical current is switched off.
- Gel is then placed in an alkaline buffer solution to denature the DNA fragments. The 2 strands of each fragment separate, exposing the bases.
8
Q
What is the southern blotting technique and how is it carried out?
A
- The strands from electrophoresis are transferred to a nitrocellulose paper or a nylon membrane, which is placed over the gel. The membrane is covered with several sheets of dry absorbent paper, drawing the alkaline solution containing DNA through the membrane by capillary action.
- The single stranded fragments of DNA are transferred to the membrane, as they are unable to pass through it. They are transferred in precisely the same relative positions as they had on the gel.
- They are then fixed in place using UV light or heated at 80 degrees.
9
Q
What is PCR?
A
Polymerase chain reaction- allows DNA to be replicated so that scientists can produce a lot of DNA from a small sample.
10
Q
What are the main steps in PCR?
A
- Separating the strands
- Annealing the primers
- Synthesis of DNA
11
Q
Explain the process of separating the strands of DNA during PCR.
A
The temperature in the PCR machine is increased to 95 decrees for 30s, this denatures the DNA by breaking the H bonds holding the strands together so they separate.
12
Q
What happens during annealing of the primers in PCR?
A
The temperature is reduced to around 55 decrees and the primers bind (anneal) to the ends of the DNA strands. They are needed for the replication of the strands to occur.
13
Q
What happens during the synthesis of DNA during PCR?
A
- The temp is increased again to 75 degrees for at least 1 minute, this is the optimum temp for Taq polymerase to work at.
- DNA polymerase adds bases to the primer, building up complementary strands of DNA and so producing double stranded DNA identical to the original sequence.
- The enzyme Taq polymerase is used, which is obtained from the thermophilic bacteria found in hot springs.
14
Q
What are the uses of DNA profiling?
A
- Forensics- PCR and DNA profiling is performed on traces of DNA left at a crime scene. The profile is then compared to that of a sample taken from a suspect or can be identified from a criminal database.
- Prove paternity- it is used when the paternity of a child is in doubt and in immigration cases to prove/disprove family relationships.
- Identify a species to which an organism belongs to.
- Used to determine the evolutionary relationships between different species.
- Identifying those who are at risk of developing particular diseases. Certain non-coding microsatellites or the repeating pattern they make, have been found to be associated with increased risk/ incidence of particular diseases, including various cancers and heart disease. Theses specific gene markers can be identifies and observed in DNA profiles.
15
Q
What is a genome?
A
All the genetic material of an organism.
16
Q
What is an exon?
A
Regions of DNA that codes for proteins.
17
Q
What is an intron?
A
Regions of non-coding DNA or RNA
18
Q
What is satellite DNA?
A
Short sequences of DNA that are repeated many times within the introns, telomeres and centromeres.
19
Q
What is a mini satellite?
A
A sequence of 20-50 base pairs repeated 50+ times, AKA variable tandem repeats.
20
Q
What is a short tandem repeat?
A
A microsatellite- a smaller region of 2-4 bases repeated 5-15 times.
21
Q
What is a histone?
A
Proteins that form a complex with DNA called chromatin
22
Q
What is hybridisation?
A
The addition of fluorescent or radioactive probes to DNA fragments via complementary base pairing.
23
Q
What are restriction endonucleases?
A
Enzymes that chop up a DNA strand into lots of small pieces.
24
Q
What is electrophoresis?
A
A type of chromatography that relies on the way charged particles move through a gel under the influence of an electrical current. Used to separate nucleic acid fragments.
25
What is a telomere?
A structure at the end of a chromosome.
26
What is a VNTR?
A variable number tandem repeat is a sequence of base pairs that is repeated 50 to several 100 times.
27
What is DNA profiling?
Producing an image of the patterns in the non-coding DNA of an individual.
28
Why is the electrical current switched off during PCR as soon as the smaller fragments reach the anode?
If left for a long period of time, the larger fragments would catch up to the smaller fragments and would produce a clustered, unreadable pattern.
29
Why is non-coding DNA used instead of coding DNA during DNA profiling?
Non-coding DNA is used as coding DNA is very similar for everyone, so it would be difficult to distinguish between different people's profiles/results.
30
What is the purpose of a DNA ladder?
It is a set of standards that are used to identify the approx size of a molecule run on a gel during electrophoresis.
31
What are the principles of DNA sequencing?
1. DNA for sequencing is mixed with primer, DNA polymerase, an excess of normal nucleotides and terminator bases.
2. Mixture is placed in a thermal cycler- a piece of equipment as used for PCR the rapidly changes temp at programmed intervals in repeated cycles- at 96 degrees the double stranded DNA separates into single strands, at 50 the primers anneal to the DNA strand.
3. At 60, DNA polymerase starts to build up new strands by adding nucleotides with the complementary bases to the single-strand DNA template.
4. Each time a terminator base is incorporated instead of a normal base, the synthesis of DNA is terminated as no more bases can be added. As the chain-terminating bases are present in lower amounts and are added at random, 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. The DNA fragments are separated according to length by capillary sequencing, on minute capillary tubes. 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.
5. The order of bases in the capillary tubes shows the sequence of the new, complementary strand of DNA which has been made. This is used to build up the sequence of the original DNA strand. The data from the sequencing process is fed into a computer the reassembles the genomes by comparing all the fragments and finding the areas of overlap between them. Once the genome is assembled, scientists want to identify the genes or parts of the genome that code for specific characteristics. Medical researchers want to identify regions that are linked with particular diseases.
32
What are the main differences of next gen sequencing?
Its automated and high throughput sequencing. It takes place on a plastic slide and replicated in situ using PCR to form clusters of identical DNA fragments instead if using a gel or capillaries. The sequencing process still adds coloured terminator bases to stop the reaction so an image can be taken. As all of the clusters are being sequenced and imaged at the same time, the technique is known as massively parallel sequencing and sometimes known as next gen sequencing.
33
What is bioinformatics?
It is the development of the software and computing tools needed to organise and analyse raw biological data, including the development of algorithms, mathematical models, and statistical tests that help us to make sense of the data being generated.
34
What is computational biology?
It uses the data from bioinformatics to build theoretical models of biological systems which can then be used to predict what will happen in different circumstances. Computational biology is the study of biology using computational techniques, especially in the analysis of huge amounts of biodata.
35
Give an example of how computational biology is used.
It is important in the analysis of the data from sequencing the billions of base pairs in DNA, for working out the 3D structures of proteins and the understanding of molecular pathways such as gene regulation.
36
What does the sequencing of the genomes of pathogens enable?
- Doctors to find out the source of an infection
- Doctors to identify antibiotic-resistant strains of bacteria, ensuring antibiotics are only used when they will be effective and helping to prevent the spread of antibiotic resistance.
- Scientists to track the progress of an outbreak of a potentially serious disease and monitor potential epidemics.
- Scientists to identify regions in the genome of pathogens that may be useful targets in the development of new drugs and to identify the genetic markers for use in vaccines.
37
What is DNA barcoding?
- It identifies particular sections of the genome that are common to all species but vary between them, so comparisons can be made.
- scientists identified species using relatively short sections of DNA from a conserved region of the genome.
38
How is DNA barcoding carried out for animals?
- a 648 base-pair section of the mitochondrial DNA in the gene cytochrome c oxidase, which codes for an enzyme involved in cellular respiration.
- This section is small enough to be sequenced quickly and cheaply, yet varies enough to give clear differences between species.
39
How can DNA barcoding be carried out on land plants?
- The region of DNA that is used in animal sequencing doesn't evolve quickly enough to show clear differences between species, but two regions in the DNA of the chloroplasts have been identified that can be used in a similar way to identify species.
40
What is a downside of DNA barcoding?
So far, scientists haven't yet been able to come up with a suitable region for fungi and bacteria, and they may not be able to.
41
In what ways has DNA barcoding been helpful to scientists?
- It can help them to understand the evolutionary relationships between organisms.
- DNA sequences of different organisms can be compared- because the basic mutation rate of DNA can be calculated, scientists can calculate how long ago 2 species diverged from a common ancestor. DNA sequencing enables scientists to build up evolutionary trees with an accuracy they haven't had before.
42
What is proteomics?
It is the study and amino acid sequencing of an organisms entire protein complex.
- The DNA sequence of the genome should, in theory, enable the prediction of sequences of the amino acids in all of the proteins it produces. The evidence is that the sequence of amino acids is not always what would be predicted from the genome sequence alone. Some genes can code for many different proteins.
43
What are spliceosomes?
The mRNA transcribed from the DNA in the nucleus includes both exons and introns. Before it lines up on the ribosomes to be translated, this 'pre-mRNA' is modified in a number of ways. The introns are removed, and in some cases, some of the exons are removed as well. Then the exons to be translated are joined together by enzyme complexes known as spliceosomes to give the mature functional mRNA.
- The spliceosomes may join the same exons in a variety of ways. As a result, a single gene may produce several versions of functional mRNA, which in turn would code for different arrangements of amino acids, giving different proteins and resulting in several different phenotypes.
44
What is synthetic biology?
An emerging area of research that can broadly de described as the design and construction of novel artificial biological pathways, organisms or devices, or the redesign of existing natural biology.
45
What techniques does synthetic biology include?
- Genetic engineering; may involve a single change in a biological pathway or relatively major genetic modification of an entire organism
- use of biological systems or parts of a biological system in industrial contexts e.g. the use of fixed or immobilised enzymes and the production of drugs from microorganisms.
- The synthesis of new genes to replace faulty genes e.g. in developing treatments for cystic fibrosis, scientists have synthesised functional genes in the lab and use them to replace the faulty genes in the cells of people affected by CF.
- They synthesis of an entire new organism
46
How do you isolate the desired gene for genetic engineering?
- most common technique is to use restriction endonucleases to cut the required gene from the DNA of an organism. Many restriction endonucleases cut the 2 strands unevenly, leaving one of the strands of the DNA fragment a few bases longer than the other strand. These regions with unpaired bases are called sticky ends. It makes it much easier to insert the desired gene into the DNA of a different organism.
- You can also isolate the mRNA for the desired gene and using the enzyme reverse transcriptase to produce a single strand of complementary DNA. The advantage of this technique is that it makes it easier to identify the desired gene, as a particular cell will make some very specific types of mRNA. e.g. beta cells of the pancreas make insulin, so produce lots of insulin mRNA molecules.
46
How is DNA inserted into a plasmid?
- The same restriction endonuclease that was used to isolate the DNA fragment is used to cut open the plasmid.
- This results in the plasmid having the complementary sticky ends to the sticky ends of the DNA fragment.
- Once the complementary bases of the 2 sticky ends are lined up, the enzyme DNA ligase forms phosphodiester bonds between the sugar and phosphate groups on the 2 strands of DNA, joining them together.
- The plasmids used as vectors are usually given a second marker gene, which is used to show that the plasmid contains the recombinant gene. This maker gene itself is often placed in the plasmid by genetic engineering.
46
Why are plasmids used as vectors for genetic modification?
They contain a maker gene. e.g. if they've been engineered to have a gene for antibiotic resistance, it enables scientists to determine that the bacteria have been taken up by the plasmid, by growing the bacteria in the media containing the antibiotic.
47
How is the marker gene inserted into the plasmid?
- The plasmid is cut by a restriction enzyme within this marker gene to inset the desired gene. If the DNA fragment is inserted successfully, the marker gene will not function.
47
Explain a method of transferring the plasmid into the host cell.
Culture bacterial cells and plasmids in a calcium rich solution and increase the temperature.
- This causes the bacterial membrane to become permeable and the plasmids can enter.
48
What is electroporation?
- Another method of transformation.
- A small electrical current is applied to the bacteria. This makes the membranes very porous and so the plasmids move into the cells.
48
What can electroporation also be used for other than transformation?
- Can get DNA fragments directly into eukaryotic cells.
- The new DNA will pass through the cell membrane and the nuclear membrane to fuse with the nuclear DNA.
- It is effective, however the current has to be carefully controlled or the membrane will be permanently damaged or destroyed, which in turn destroys the whole cell.
49
What is electrofusion?
- Tiny electric currents are applied to the membranes of 2 different cells. This fuses the cell and nuclear membranes of the 2 different cells together to form a hybrid or polyploid cell, containing DNA from both.
50
What are the pros and cons of electrofusion?
- It is used differently in animal cells as they don't fuse as easily and effectively as plant cells.
- It is useful for making GM plants.
- It is useful in producing monoclonal antibodies.
51
Why are GM microorganisms used to store a living record of the DNA of other organisms in DNA libraries?
- DNA sequencing projects e.g. the HGP, enable scientists to build a collection of sequenced DNA fragments from one organism that is then stored in microorganisms throughout the process of genetic engineering.
- These libraries serve as a source of DNA fragments for further genetic engineering applications or for further study of their functions.
51
What are the ethical concerns of genetically modifying microorganisms?
- Initially people were uncomfortable with inserting human genes into microorganisms but the pure human medicines, antibiotics and enzymes produced are now seen as overwhelmingly beneficial.
- They have been safely used for many years, so there is relatively little ethical debate about the use of GM microorganisms except for the manipulation of pathogens in biological warfare.
52
What are the risks and benefits of genetically engineering pest resistance?
Pros;
- Pest-resistant GM crop varieties reduce the amount of pesticide spraying, protecting the environment and helping poorer farmers.
- Increased yield
Cons;
- Non-pest insects and insect-eating predators might be damaged by the toxins in the GM plants
- Insect pests may become resistant to pesticides in GM crops
53
What are the risks and benefits of genetically engineering disease resistance?
Pros;
- Crop varieties resistant to common plant diseases can be produced, reducing crop losses/ increasing yield.
Cons;
- Transferred genes might spread to wild populations and cause problems e.g. superweeds
54
What are the risks and benefits of genetically engineering herbicide resistance?
Pros;
- herbicides can be used to reduce competing weeds and increase yield
Cons;
- Biodiversity could be reduced if herbicides are overused to destroy weeds.
- Fear of superweeds
55
biology
flashcards
Decks in class (13)
# Cards
-
13- neuronal communication79
-
14- hormonal communication35
-
15 - homeostasis38
-
15.4-15.8 Excretion, liver and kidney41
-
16- plant responses32
-
17- energy for biological processes41
-
18 - respiration20
-
19- genetics of living systems25
-
20- patterns of inheritance and variation22
-
21- manipulating genomes60
-
22- cloning and biotech60
-
23- ecosystems54
-
24 - populations and sustainability51
