6.1.3 Manipulating genomes

Question 1

What is the purpose of PCR?

Answer 1

used to amplify fragments of DNA, generating millions of copies from a small DNA sample
used in forensic science

Question 2

What four components are needed for PCR?

Answer 2

-DNA frangments
-free DNA nucleotides
-primers
-DNA/Taq polymerase, extracted from thermophilic bacteria

Question 3

describe the function of pimers

Answer 3

short pieces of DNA
they bind to the beginning of DNA fragments and initiate replication

Question 4

describe the three stages of PCR

Answer 4

1. The mixture is heated to 95°C, breaking H bonds between DNA strands
2. The mixture is cooled to around 60°C, allowing primers to anneal to the DNA
3. Temperature increased to 72°C, optimum temperature for Taq polymerase to build the complementary strands of DNA

Question 5

How many cycles of PCR are typically carried out?

Answer 5

Around 30-40 cycles, each doubling the amount of DNA, generating millions of copies.

Question 6

What is the purpose of electrophoresis in DNA analysis?

Answer 6

Used to separate DNA fragments based on their size so they can be analysed

Question 7

Why is a fluorescent molecule added to DNA before electrophoresis?

Answer 7

Fluorescent dye binds to DNA and makes it visible under UV light.

Question 8

Name a common fluorescent tag

Answer 8

Ethidium bromide

Question 9

How does gel electrophoresis separate DNA fragments?

Answer 9

DNA is negatively charged, so when placed in an electric field, it moves toward the positive electrode (anode).

Question 10

Which DNA fragments will move the quickest towards the anode?

Answer 10

Shorter DNA fragments travel faster and a longer distance than larger fragments

Question 11

What type of gel is used in electrophoresis?

Answer 11

Agorose gel

Question 12

What are the key steps in gel electrophoresis?

Answer 12

1. Prepare agarose gel with wells at the cathode side.
2. Place gel into a tank with buffer solution that conducts electricity.
3. Mix DNA with loading dye to make it darker & visible.
4. Pipette a fixed volume of the DNA samples into the wells.
5. Run an electric current, so DNA moves towards the anode.
6. When dye has reached the bottom, turn off electrcity
7. Visualise banding pattern under UV light.

Question 13

How would electrophoresis help in forensics?

Answer 13

Compare the banding pattern with other DNA samples

Question 14

What are restriction enzymes?

Answer 14

Enzymes that cut double-stranded DNA at specific recognition sequences.

Question 15

What is a recognition sequence?

Answer 15

A specific DNA sequence where a restriction enzyme cuts. It reads the same forwards and backwards on complementary strands.

Question 16

What are the two types of DNA ends produced by restriction enzymes?

Answer 16

Blunt ends – DNA is cut straight down the middle.
Sticky ends – DNA is cut in a zig-zag, leaving single-stranded overhangs that can pair with complementary sequences.

Question 17

How do restriction enzymes cut DNA?

Answer 17

They cut double-stranded DNA by hydrolysing the phosphodiester backbone

Question 18

Why are sticky ends useful in genetic engineering?

Answer 18

Sticky ends can easily bind to complementary DNA sequences, making it easier to insert DNA fragments into vectors like plasmids.

Question 19

What is DNA profiling?

Answer 19

A technique used to analyse and compare DNA samples to identify individuals or determine genetic relationships.

Question 20

What is the first step in DNA profiling?

Answer 20

Collect DNA sample and amlify it using PCR

Question 21

List 4 uses of DNA profiling

Answer 21

-forensic science
-paternity testing
-determining evelotionary relationships
-analysis of disease risk

Question 22

What is genetic engineering?

Answer 22

Modification of an organism’s genome by inserting, deleting, or altering DNA sequences to produce desired traits.

Question 23

How is insulin produced using genetically engineered bacteria?

Answer 23

1. The insulin gene is cut from human DNA using restriction enzymes.
2. A plasmid is cut with the same enzyme.
3. DNA ligase joins the complementary sticky ends, forming recombinant DNA.
4. The recombinant plasmid is inserted into bacteria using electroporation.
5. The bacteria multiply and produce insulin, which is then extracted.

Question 24

How does an electroporator help with genetic engineering?

Answer 24

It creates an eletric field
To make the bacterial membrane more permeable

Question 25

What is the word for bacteria that have had their DNA modified to possess and express an extra (foreign) gene?

Answer 25

Transgenic bacteria

Question 26

What is the role of restriction enzymes in genetic engineering?

Answer 26

Cut DNA at specific recognition sites, creating sticky or blunt ends for recombining DNA.

Question 27

How are genetically modified animals used in drug production?

Answer 27

1. The gene for a therapeutic protein is inserted into a fertilised animal egg. 2. The embryo is implanted into a surrogate animal. 3. The animal produces the drug in its milk, which can be extracted and purified.

Question 28

List the 6 arguments **for** the use of GMOs

Answer 28

1. Increase crop yield and make food more nutritious 2. Farmers can savemoney on pesticides and environmental issues associated with pesticides are reduced 3. No allegic reactions 4. Vaccines using GM plants do not need to be kept cold, so remote regions can use them 5. Enzymes can be produced using GMOs often used in industrial processes, e.g washing detergents 6. Cheap as once one is made many more can be made, making drugs cheaper

Question 29

List 5 arguments **against** the use of GMOs

Answer 29

1. GMOs are often patented and seeds are expensive to buy-some places are at a disadvantage 2. Cross-pollination can occur, may cause herbicide-resistant plants 3. Ethical issues-should humans manipulate animals for their benefit 4. Religious reasons-no consent given 5. People may worry about the long term impact

Question 30

What is gene therapy used for?

Answer 30

Used to treat genetic diseases by introducing a functional copy of a gene into a patient’s cells.

Question 31

How does gene therapy work for recessive diseases?

Answer 31

A functional copy of the gene is introduced into the patient’s cells using a **vector**, allowing the synthesis of the missing protein.

Question 32

How does gene therapy work for dominant diseases?

Answer 32

A piece of DNA is inserted into the mutated allele to silence it and prevent its expression.

Question 33

What is germline gene therapy?

Answer 33

A functional allele is inserted into a gamete. It is passed onto offspring Currently illegal

Question 34

What is somatic gene therapy?

Answer 34

A functional allele is inserted into the body cells that are affected by the disorder. Only changes some cells and needs to repeated when those cells eventually die

Question 35

List the problems associated with gene therapy

Answer 35

1. For somatic, the gene would need to be insterted multiple times 2. The body may see the vector as foreign and trigger an immune response, reject it 3. May be inserted in the wrong place=harmful effects 4. Expensive 5. For germline, future generations may inherit - issues of consent 6. Ethical issues - used in other areas, like cosmetics

Question 36

What is the purpose of the chain-termination/Sanger sequencing method?

Answer 36

It determines the sequence of DNA bases by creating DNA fragments of varying lengths and reading the terminal base of each fragment.

Question 37

What are the key components needed for the chain-termination method?

Answer 37

-DNA sample -Free nuleotides -Primers -DNA polymerase -Fluorescently-labelled modified nucleotides

Question 38

What happens when a fluorescently-labelled modified nucleotide is added to the DNA chain?

Answer 38

It prevents further nucleotide addition, creating DNA fragments of varying lengths.

Question 39

What happens in the first stage of Sanger sequencing?

Answer 39

The DNA sample, DNA polymerase, free nucleotides and fluorescently-labelled modified nucleotides are mixed in 4 small tubes. Each fluorescent base is added to one test tube.

Question 40

Describe the second stage of Sanger sequencing

Answer 40

PCR is carried out

Question 41

How are the DNA fragments separated?

Answer 41

Electrophoresis

Question 42

How is the DNA sequence determined from the gel?

Answer 42

-The smallest fragment ( i.e one base pair) is at the bottom of the gel. -The sequence is read from bottom to top

Question 43

What are the 3 more advanced ways of sequencing?

Answer 43

-Automated sequencing, putting all 4 bases in the same tube -High throughput sequencing -Pyrosequencing

Question 44

DNA profiling makes use of the fact that DNA varies between individuals. When creating a DNA profile, DNA is first extracted from a sample of tissue. Outline the subsequent steps involved in producing a DNA profile. (4)

Answer 44

1. Amplify (DNA fragment) with PCR 2. Cut / digest , with restriction enzyme 3. Separate , using gel electrophoresis / electric current through gel 4. Transfer fragments to membrane nylon (southern blotting) 5. Add , radioactive / fluorescent , probe 6. Use , x-rays / UV light , to view position of DNA fragments.

Question 45

DNA codes for proteins within the cell. Some regions of DNA are described as non-coding. (i) Explain why some regions of DNA can be described as ‘non-coding’. (2)

Answer 45

* Editing of primary , mRNA / transcript * Not present in mature mRNA * Not translated * Regulatory , sequences / genes