T7 - Modern Genetics

Question 1

State what is meant by the term genome.

Answer 1

All the DNA within an organism including nuclear, mitchondrial and chloroplast DNA.

Question 2

What does PCR stand for?

Answer 2

Polymerase chain reaction

Question 3

What is the purpose of PCR?

Answer 3

To make large numbers of copies of DNA fragments.

Question 4

How are samples of DNA amplified by PCR used?

Answer 4

● to predict the amino acid sequence of proteins and possible links to genetically determined conditions, using gene sequencing.
● in forensic science, to identify criminals and to test paternity, using DNA profiling.
● in taxonomy, to determine the evolutionary relationshop between species of organism.

Question 5

What is required for PCR?

Answer 5

DNA sample + Taq polymerase + primers + nucleotides + pH buffer > PCR machine (thermal cycler).

Question 6

Describe the steps in PCR

Answer 6

1. Put DNA sample + Taq polymerase + primers + nucleotides + pH buffer into PCR machine (thermal cycler).
2. Denature DNA sample at 90-95c for 30s to separate the two strands (breaks the hydrogen bonds between bases)
3. Cool sample to 50-55c to allow primers to anneal (bind) to DNA strand.Elongate (copy)
   4.Heat to 75c (optimum temperature for Taq polymerase) so Taq polymerase extends the nucleotide chain from the primers (therefore
   primers are used to select the sequence to be copied).
4. Repeat for 30 cycles (creates over 1 billion copies).

Question 7

Explain the need to heat the DNA to 90-95oC

Answer 7

High heat is needed to break the hydrogen bonds between the bases of the two strands of DNA, so they separate.

Question 8

Describe the role of the primers in PCR.

Answer 8

Two primers are designed to select the sequence of DNA you want to copy.
Through complementary base pairing, one primer attaches to the top strand at one end of the sequence of interest, and the other primer attaches to the bottom strand at the other end.
Primers also act as a starting point for Taq polymerase.

Question 9

Explain why the enzyme Taq polymerase from the thermophilic bacterium Thermus aquaticus, which is found in hot springs, used instead of eukaryotic DNA polymerase?

Answer 9

The enzyme’s optimum temperature (72oC) is much higher than most organisms, so it is able to function at the high temperatures used in PCR without denaturing.

Question 10

A single cycle of PCR takes 5 minutes. Calculate the number of DNA fragments produced by PCR:
i) 20 min
ii) 1 hour
iii) 3 hours

Answer 10

i) 20/5 = 4. 2^4 = 16
ii) 60/5 = 12. 2^12 = 4096
iii) 180/5 = 36. 2^36 = 6.9 x 10^10

Question 11

Describe possible sources of contamination in prepareing a PCR sample

Answer 11

from microbes in the environment
from dirty equipment
from the researcher

Question 12

Describe suitable precautions the reseacher can take to reduce the risk of contamination

Answer 12

● the research should wear hair net, gloves, lab coat to prevent their DNA contaminating the sample.
● clean work surfaces, use sterile solutions, and disposable equipment.

Question 13

State the differences between introns and exons

Answer 13

Introns are non-coding regions of DNA, whereas as exons are coding regions of DNA.
Introns are spliced out of mRNA before it leaves the nucleus, whereas exons remain in mature mRNA.
Introns make up >90% of DNA

Question 14

What part of DNA is used in DNA profiling?

Answer 14

Introns

Question 15

Describe the short sequences of DNA that are repeated many times within an intron

Answer 15

Mini-satellites (variable number tandem repeats) are 10-100 base sequences repeated 50 to several hundred times.
Micro-satellites (short tandem repeat) are 2-6 base sequence repeated 5 to 100 times.

Question 16

Describe how a DNA profile is produced.

Answer 16

Micro- and mini- satellites are cut out with specific restriction enzymes.
The restriction enzymes will cut either side of the satellites at specific recognition sites, leaving fragments of DNA that are different sizes depending on how many repeats are in the different satellites.
The fragments are then separated using an electric current by gel electrophoresis, which produces bands at different distances along the gel depending on the size of the fragments.

Question 17

Explain why everyone’s DNA profile is unique

Answer 17

The same micro or mini satellite appears in the same place on each homologous chromosome, but the number of repeats of each satellite will vary as different patterns may be inherited from your father and mother. There are many different introns, and huge variation in the number of repeats, so the likelihood of two people having same pattern in DNA is remote, except for identical twins.

Question 18

Name two enzymes required to produce recombinant DNA

Answer 18

Restriction (endonuclease) enzymes
DNA ligase

Question 19

What is the function of restriction enzymes?

Answer 19

Cut DNA at specific reognition sites, and leave ‘sticky ends’

Question 20

What is the functon of DNA ligase?

Answer 20

Catalyses a condensation reaction which makes phosphodiester bonds between nucleotides

Question 21

Describe how a recombinant plasmid is produced.

Answer 21

The plasmid and gene of interest are isolated and cut using the same ‘sticky end’ restriction enzyme.
The ends of the gene and plasmid are joined using DNA ligase, which joins nucleotides together using phosphodiester bonds.

Question 22

Describe how to produce large numbers of genetically modified bacteria

Answer 22

Isolate the foreign gene and bacterial plasmid.
Use the same restriction enzyme to cut out gene and plasmid at specific recognition site, forming sticky ends.
Use (DNA) ligase to insert gene into plasmid.
Transform the bacteria by inserting recombinant plasmid.
Culture the transformed bacteria in optimal growth condition.

Question 23

How can liposomes be used to insert recombinant DNA into eukarytoic cells?

Answer 23

Liposome wrapping wraps recombinant DNA in a phospholipid bilayer, which fuses with cell membrane.
They can be targeted to specific cell types by placing specific receptors on their surface.

Question 24

Desribe the advantages of using viruses to to insert recombinant DNA into a host.

Answer 24

Viruses can integrate their DNA directly into the host’s genome.
Different viruses insert the DNA in different eays so scientists can control where and for how long the DNA is expressed.

Question 25

Desribe the disadvantags of using viruses to to insert recombinant DNA into a host.

Answer 25

Virsues can only carry very small pieces of DNA, and there may be unexpected side effects depending on where in the host’s chromsome the DNA integrates itself.

Question 26

Name three other ways of transferring DNA

Answer 26

Gene guns, microinjection, electroporation (electric field makes pores in membrane)

Question 27

Explain how antibiotic resistance marker genes and replica plating are used to identify recombinant cells.

Answer 27

The foreign gene is inserted into an antibiotic resistance gene in plasmid, disrupting it.
Transformed bacteria will therefore not be resistant to this antibiotic.
Replica plating transfers colonies of bacteria from a master plate onto a plate containing the antibiotic using sterile velveteen.
Colonies grown from transformed bacteria will not survive on this plate.
Replica plating keeps a living sample of the pattern of colonies without treatment.

Question 28

Describe how to carry out replica plating

Answer 28

Press sterile velveteen gently onto the colonies of bacteria on the master plate.
Then, keeping the same orientation, press lightly onto another plate.
Repeat with other plates.

Question 29

Why are mice good model organisms?

Answer 29

They share many genes with humans
Mice with nonfunctioning genes can be bred and characteristics observed in short space of time.

Question 30

What are gene knockout mice?

Answer 30

Genetically modified mice that have had an existing gene of interest inactivated, or “knocked out,” by replacing it or disrupting it with an artificial piece of DNA.

Question 31

How is recombinant DNA inserted in plants such as Soya beans?

Answer 31

A Ti is isolated from Agrobacterium along with DNA containing gene of interest from donor organism.
The gene is cut out using the same ‘sticky end’ restriction enzyme used to cut open the Ti plasmid.
The sticky ends of the gene and Ti plasmid are joined using DNA ligase.
Agrobacterium is transformed by reinserting recombinant Ti plasmid.
Agrobacterium is used as a vector to carry foreign gene into the plant cell’s genome.
An explant of infected cells is taken and tissue culture used to produce plants in which all cells have foreign gene.

Question 32

Explain why genetic modifcation of Soya bean plants has been used to improve production of Soya beans?

Answer 32

Genetically modified Soya produces more oleic acid and less linoleic acid.
This is good because linoleic acid oxidises easily causing soya oil to be unusable, whereas oleic acid is not easily oxidised, giving products a longer shelf life.

Question 33

Describe the advantages of genetic modification

Answer 33

Increase crop yields, nutritional value, shelf life.
Decreases use of pesticides, herbicides, animal remedies.
Produces drought resistant, salt tolerant crops.
Leads to improved human health and medicines.

Question 34

Describe the disadvantages of genetic modification

Answer 34

Possible spread of transgenes into other species.
Animal welfare and ethical issues; animals may suffer shortened life spans.
GMOs may cause emergence of resistance in insects/pests.
Developing countries may become dependent on GM technology of developed countries