Manipulating genomes

Question 1

What is a genome?

Answer 1

Of an organism is all of the genetic material it contains.

Question 2

Producing a DNA profile has how many stages?

Answer 2

5

Question 3

Introns -

Answer 3

Large non-coding regions of DNA that are removed from the mRNA before it transferred to the polypeptide chain.

Question 4

Producing a DNA profile step 1:

Answer 4

1. Extracting the DNA where the DNA is extracted from a tissue sample.

Question 5

Producing a DNA profile step 2:

Answer 5

Digesting the sample -
2. The strands of DNA are cut into smaller fragments using special enzymes called restriction endonucleases, these cut DNA at a specific nucleotide sequence. Allow scientists the ability to cut the DNA at defined points in the introns.

Question 6

Producing a DNA profile step 3:

Answer 6

Separating the DNA fragments -
3. The cut fragments of DNA need to be separated to form a clear and recognisable pattern this is done by using electrophoresis a gel is used to in order to separate the DNA double strands into single strands.

Question 7

Producing a DNA profile step 4:

Answer 7

Hybridisation -
4. DNA probes are added to the label the fragments where these radioactive probes attach to specific fragments, these DNA probes are short DNA or RNA sequences which are complementary to the known DNA sequence which bind to complementary strands under conditions of pH and temperature.

Question 8

Producing a DNA profile step 5:

Answer 8

Seeing the evidence
5. If radioactive labels were added to the DNA probes, X-ray images are taken of the paper if they use fluorescent labels the paper is placed under UV light, the fragments give a pattern of bars which is unique to all individuals except identical siblings. Can be used to see how many fragments were inherited from each parent.

Question 9

Separation of nucleic acids fragments by electrophoresis -

Answer 9

DNA fragments are put into walls in agarose gel strips which contain a buffer to maintain a pH.
When an electric current is passed through the electrophoresis plate the DNA fragments move through the gel to the positive anode because of the negatively charged phosphate groups. The rate of movement depends on mass or length of DNA fragments, smaller fragments overtime are easier to move than larger fragments.

Question 10

Polymerase chain reaction -

Answer 10

DNA profiling is often used in forensics and only limited samples are available, PCR is used where DNA is replicated and allows a lot of DNA from the tiniest original sample.

The DNA to be amplified, the smaller primer DNA sequences and the enzyme DNA polymerase are mixed together and placed in a PCR machine where the temperature is carefully controlled at intervals, the reaction can be repeated about 30 give a billion copies.

Question 11

PCR steps 1,2 and 3 -

Answer 11

1. Separating the strands - temperature is increased and this denatures the strands by breaking the hydrogen bonds holding the strands
2. The temperature is decreased from around 90 degrees to around 50 degrees and the primers bind to the ends of the DNA strands they are needed for replication
3. The temperature is increased to around 70 degrees which is the optimum for DNA polymerase to occur and allows the synthesis of the primer building up complementary strands of DNA which is identical to the original.
4. These strands can then be re-used in the polymerase chain reaction.

Question 12

Uses of DNA profiling -

Answer 12

Forensics and disease risk are the most common uses:

Forensic science -
PCR and DNA profiling can be used and performed on traces of DNA left at a crime scene such, the DNA profile is compared to a potential suspect

Family issues -
Can be used for the proof of paternity of a child when in doubt. Allows a proof of family relationships.

Risks of developing disease -
Specific gene markers can be identified and observed in DNA profiles and have been found to be associated with particular diseases including such cancers.

Question 13

Principles of DNA sequencing brief -

Answer 13

DNA is chopped into fragments and each fragment is sequenced. Involves terminator bases which are modified versions of the 4 nucleotide bases.

Question 14

DNA sequencing step 1:

Answer 14

The DNA for sequencing is mixed with a primer, DNA polymerase excess of normal nucleotides containing A,T,C,G and terminator bases.

Question 15

DNA sequencing step 2 :

Answer 15

Mixture is placed in a thermal cycler, like in PCR, rapidly changing temperatures are similar to what is seen in PCR where at 60 degrees DNA polymerase builds new DNA strands by adding nucleotides with the complementary base to the single-strand DNA template.

Question 16

What happens each time a terminator base is incorporated in DNA sequencing?

Answer 16

The synthesis of DNA is terminated and no more bases can be added, results in many fragments of different lengths, these DNA fragments are separated according to their length by capillary sequencing like gel electrophoresis.

Question 17

What does the final step show in DNA sequencing?

Answer 17

The order of bases in the capillary tube show the new complementary strand of DNA which has been made and which builds up the sequence of original DNA, the data of sequencing is fed into a computer.

Question 18

Stages summarised of DNA sequencing 1-5 -

Answer 18

1/2. DNA chopped up and is mixed with primers, bases, DNA polymerase and terminator bases

3. Each time a terminator base is added a strand terminates until all possible chains produce
4. Readout from the capillary tubes DNA fragments are separated by electrophoresis in capillary tubes.
5. Computer analysis of all data to give original DNA sequence

Question 19

Analysing the genome can be used from doctors in from understanding the such pathogens -

Answer 19

• Doctors can find out sources of infection and track the progress of an outbreak as an example.

Question 20

Identifying species (DNA barcoding) -

Answer 20

Identifying species can be achieved from relatively short sections of DNA from a conserved regions of DNA for example some codes are only representative of some organisms.

Question 21

Evolutionary relationships -

Answer 21

Given scientists powerful tools, DNA of different organisms can be compared and build up evolutionary trees.

Question 22

Synthetic biology is a result of the use of DNA sequencing what is synthetic biology?

Answer 22

Design and construction of novel artificial biological pathways, organisms or devices or the redesign of existing natural biological system.

Question 23

Uses of synthetic biology -

Answer 23

Genetic engineering
Use of fixed and the production of drugs from microorganisms. Synthesis of new genes to replace faulty ones

Question 24

Genetic engineering -

Answer 24

Manipulation of a genome, involves isolating a desired gene with the desirable characteristic in one organism, use a suitable vector and they become a GMO.

Question 25

Step 1 of genetic engineering is?

Answer 25

Isolate the desired gene. Most common technique uses enzymes called restriction endonucleases which is restricted to breaking specific DNA strands at base sequences.

Question 26

Formation of recombinant DNA -

Answer 26

The DNA strand isolated by the restriction endonucleases must be inserted into a vector that can carry into a host cell.

Question 27

Vectors used in genetic engineering -

Answer 27

Most common ones used in genetic engineering are bacterial plasmids. Once inside a host cell it can combine with the hosts DNA to form what is called a recombinant.

Question 28

How to insert a DNA fragment in a plasmid used as vector -

Answer 28

Must be cut open first, the same restriction endonucleases are used to first cut open the plasmid results in complementary sticky ends to the sticky ends of the DNA fragment once formed DNA ligase forms phosphodiester bonds between the sugar and the phosphate group.

Question 29

Transferring the vector/plasmid -

Answer 29

The plasmid with the recombinant DNA must be transferred to the host cell in transformation.
One method is electroporation where a small electrical current is applied making the membranes very porous and so plasmids can move in.

Can also be used in eukaryotic cells where the new DNA will pass over the membrane and fuse with the nuclear DNA.

Question 30

Ethical positives of GM plants -

Answer 30

It is thought that the genetic modification of plants will help the feed the ever-growing population and overcome carbon issues.

Question 31

Pros of GM cropping -

Answer 31

Pest resistance crops reduce the amount of pesticides feeding and increase crop yield. Herbicide resistance crops. Extended shelf life and reduces food waste can also offer nutritional value of crops can be used for human medicines

Question 32

Negatives of GM cropping -

Answer 32

Biodiversity can be reduced if herbicides are overused to destroy weeds. Extended shelf life may reduce the demand for crops. Transferred genes may be spread to wild populations and cause such problems. The nutritional value may result in allergies in some.

Question 33

GM in animals -

Answer 33

Much harder to produce GM vertebrates especially mammals.

Question 34

Ethical issues of GM animals -

Answer 34

Is it right to be human genes in animals?

Question 35

Gene therapy in humans may be needed due to diseases -

Answer 35

Such genes result in the gain of such diseases like haemophilia. They can remove such healthy genes and place them in the faulty genes of individuals. Two types of are somatic cell gene therapy and germ line cell gene therapy.

Question 36

Somatic cell gene therapy -

Answer 36

• Involves replacing the mutant allele with the healthy allele
• Getting engineered plasmids into the nucleus of the cells and will result in the expression of the healthy allele
• However, the healthy allele will be passed on every time a cell divides by mitosis but these somatic cells have a limited time capacity and are replaced from stem cells which have the faulty allele and in addition the treated individual will still pass the faulty allele onto children if they have any.

Question 37

Germ line cell gene therapy -

Answer 37

Alternative way of treating which involves inserting a healthy gene into germ cells usually the eggs or a part of IVF treatment which is lab based where the individual would be born healthy with a normal allele and would pass on healthy offspring. It is illegal in most for human embryos in most countries as there may be implications on the embryo which are unknown. Human rights of the unborn individual could be violated as there was no choice it may be further unethical as parents can essentially choose their offspring.