21- manipulating genomes

Question 1

What are the 5 main stages of DNA profiling?

Answer 1

1. extracting the DNA
2. Digesting the sample
3. separating the DNA sample
4. Hybridisation
5. Viewing results.

Question 2

Explain the first step of DNA profiling, Extracting the DNA.

Answer 2

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.

Question 3

Explain step 2 of DNA profiling- digesting the sample.

Answer 3

• 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.

Question 4

Explain the third step in DNA profiling- Separating DNA fragments.

Answer 4

• 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.

Question 5

Explain the 4th step in DNA profiling- Hybridisation.

Answer 5

• 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.

Question 6

Explain stage 5 of DNA profiling- Viewing the evidence.

Answer 6

• 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.

Question 7

Explain the process of separation of nucleic acid fragments by electrophoresis.

Answer 7

• 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.

Question 8

What is the southern blotting technique and how is it carried out?

Answer 8

• 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.

Question 9

What is PCR?

Answer 9

Polymerase chain reaction- allows DNA to be replicated so that scientists can produce a lot of DNA from a small sample.

Question 10

What are the main steps in PCR?

Answer 10

1. Separating the strands
2. Annealing the primers
3. Synthesis of DNA

Question 11

Explain the process of separating the strands of DNA during PCR.

Answer 11

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.

Question 12

What happens during annealing of the primers in PCR?

Answer 12

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.

Question 13

What happens during the synthesis of DNA during PCR?

Answer 13

• 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.

Question 14

What are the uses of DNA profiling?

Answer 14

1. 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.
2. Prove paternity- it is used when the paternity of a child is in doubt and in immigration cases to prove/disprove family relationships.
3. Identify a species to which an organism belongs to.
4. Used to determine the evolutionary relationships between different species.
5. 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.

Question 15

What is a genome?

Answer 15

All the genetic material of an organism.

Question 16

What is an exon?

Answer 16

Regions of DNA that codes for proteins.

Question 17

What is an intron?

Answer 17

Regions of non-coding DNA or RNA

Question 18

What is satellite DNA?

Answer 18

Short sequences of DNA that are repeated many times within the introns, telomeres and centromeres.

Question 19

What is a mini satellite?

Answer 19

A sequence of 20-50 base pairs repeated 50+ times, AKA variable tandem repeats.

Question 20

What is a short tandem repeat?

Answer 20

A microsatellite- a smaller region of 2-4 bases repeated 5-15 times.

Question 21

What is a histone?

Answer 21

Proteins that form a complex with DNA called chromatin

Question 22

What is hybridisation?

Answer 22

The addition of fluorescent or radioactive probes to DNA fragments via complementary base pairing.

Question 23

What are restriction endonucleases?

Answer 23

Enzymes that chop up a DNA strand into lots of small pieces.

Question 24

What is electrophoresis?

Answer 24

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.

Question 25

What is a telomere?

Answer 25

A structure at the end of a chromosome.

Question 26

What is a VNTR?

Answer 26

A variable number tandem repeat is a sequence of base pairs that is repeated 50 to several 100 times.

Question 27

What is DNA profiling?

Answer 27

Producing an image of the patterns in the non-coding DNA of an individual.

Question 28

Why is the electrical current switched off during PCR as soon as the smaller fragments reach the anode?

Answer 28

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.

Question 29

Why is non-coding DNA used instead of coding DNA during DNA profiling?

Answer 29

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.

Question 30

What is the purpose of a DNA ladder?

Answer 30

It is a set of standards that are used to identify the approx size of a molecule run on a gel during electrophoresis.

Question 31

What are the principles of DNA sequencing?

Answer 31

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.

Question 32

What are the main differences of next gen sequencing?

Answer 32

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.

Question 33

What is bioinformatics?

Answer 33

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.

Question 34

What is computational biology?

Answer 34

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.

Question 35

Give an example of how computational biology is used.

Answer 35

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.

Question 36

What does the sequencing of the genomes of pathogens enable?

Answer 36

• 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.

Question 37

What is DNA barcoding?

Answer 37

• 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.

Question 38

How is DNA barcoding carried out for animals?

Answer 38

• 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.

Question 39

How can DNA barcoding be carried out on land plants?

Answer 39

• 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.

Question 40

What is a downside of DNA barcoding?

Answer 40

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.

Question 41

In what ways has DNA barcoding been helpful to scientists?

Answer 41

• 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.

Question 42

What is proteomics?

Answer 42

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.

Question 43

What are spliceosomes?

Answer 43

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.

Question 44

What is synthetic biology?

Answer 44

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.

Question 45

What techniques does synthetic biology include?

Answer 45

• 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

Question 46

How do you isolate the desired gene for genetic engineering?

Answer 46

• 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.

Question 46

How is DNA inserted into a plasmid?

Answer 46

• 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.

Question 46

Why are plasmids used as vectors for genetic modification?

Answer 46

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.

Question 47

How is the marker gene inserted into the plasmid?

Answer 47

• 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.

Question 47

Explain a method of transferring the plasmid into the host cell.

Answer 47

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.

Question 48

What is electroporation?

Answer 48

• 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.

Question 48

What can electroporation also be used for other than transformation?

Answer 48

• 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.

Question 49

What is electrofusion?

Answer 49

• 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.

Question 50

What are the pros and cons of electrofusion?

Answer 50

• 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.

Question 51

Why are GM microorganisms used to store a living record of the DNA of other organisms in DNA libraries?

Answer 51

• 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.

Question 51

What are the ethical concerns of genetically modifying microorganisms?

Answer 51

• 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.

Question 52

What are the risks and benefits of genetically engineering pest resistance?

Answer 52

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

Question 53

What are the risks and benefits of genetically engineering disease resistance?

Answer 53

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

Question 54

What are the risks and benefits of genetically engineering herbicide resistance?

Answer 54

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