Manipulating Genomes

Question 1

In PCR, what is the importance of each ingredient: target DNA, DNA primers, DNA nucleotides, thermostable DNA polymerase, buffer, Mg2+?

Answer 1

Target DNA: contains sequence to be copied - template
DNA primers: so DNA polymerase can bind
DNA nucleotides: to extend the chain
Thermostable DNA polymerase: catalyses joining of adjacent nucleotides + is heat stable to withstand thermocycling
Buffer sol: maintain pH of solution for DNA polymerase
Mg2+: cofactor of the enzyme

Question 2

What enzyme is used to cut open the vector DNA?

Answer 2

Restriction endonuclease

Question 3

What type of bond does restriction endonuclease have to break?

Answer 3

Phosphodiester

Question 4

What are sticky ends?

Answer 4

From small sections of single stranded DNA that have been cut unequally

Question 5

What type of bond forms between complementary sticky ends?

Answer 5

Hydrogen

Question 6

What type of enzyme is used to join the gene to the vector DNA?

Answer 6

DNA ligase

Question 7

What type of bond does DNA ligase form?

Answer 7

Phosphodiester

Question 8

Define gene therapy.

Answer 8

Inserting a functional allele for a particular gene into a cell which only contains mutated/non-functioning alleles for that gene

Question 9

Outline 2 differences between germline and somatic gene cell therapy.

Answer 9

Germline: inherited, all cells are affected
Somatic: not inherited, only specific cells are affected

Question 10

Name 2 methods for delivering alleles into cells in gene therapy.

Answer 10

Virus vector and liposomes

Question 11

Gene therapy is not yet a successful technique. Outline 2 issues with both of the delivery methods.

Answer 11

Virus may cause an immune response or immunity is developed. Little control - allele not into nucleus or gene inserted in the wrong place in the genome

Question 12

Explain how the insertion of a functioning allele in gene therapy can disrupt the expression of another gene in the cell.

Answer 12

The virus may insert the gene into the middle of the sequence for another gene, causing a frame shift. It could also be inserted into a promoter region, turning genes on/off.

Question 13

Define DNA sequencing.

Answer 13

The process used to determine the precise sequence of nucleotides in a length of DNA.

Question 14

What is Sanger sequencing?

Answer 14

Chain termination. Radioactive, modified nucleotides stop DNA synthesis and single stranded acts as a template. Add DNA polymerase, free nucleotides and primers.
The strands are separated by length, and sequence read shortest to longest.

Question 15

What is a primer?

Answer 15

A short DNA strand that is complementary to the start of a DNA template.

Question 16

What is pyrosequencing?

Answer 16

Single DNA strand complementary to the sequenced strand is synthesised one base at a time. Light is generated - detect presence and intensity.

Question 17

What are the uses of DNA sequencing?

Answer 17

Genetic diseases
Synthetic biology
Bioinformatics
Predict aa sequence
Genetic relationships

Question 18

Define tandem repeats.

Answer 18

Short, repeated sequences - not code for a protein. In non coding regions.

Question 19

What are the uses of synthetic biology?

Answer 19

Develop artificial biological devices and organisms. Produce medicines - change/add/delete DNA or insert gene expressing drug/protein.

Question 20

How does DNA fingerprinting work?

Answer 20

Produce specific pattern of bands from tandem repeats in individual genome. Restriction enzymes used to cut DNA backbone.

Question 21

What is needed for PCR and what is the main function?

Answer 21

Function: amplifies DNA fragments outside living organism.
Thermostable DNA polymerase comes from thermophilic bacteria = extremophiles and not denature at high temps.

Question 22

What are the steps of PCR?

Answer 22

1. Denaturation at 95’ hydrogen bonds break and strands separate
2. Annealing at 68’ primers bind to 3’ end of DNA and mark sites for amplification
3. Elongation at 72’ DNA polymerase adds nucleotides to 3’ end of primer

Amplify fragments before sequencing.

Question 23

What are the uses of PCR?

Answer 23

• identify viral infections
• forensic science
• evolutionary relationships research
• tissue typing
• detect mutations
• detect oncogenes

Question 24

What is the main function of electrophoresis and how does it work?

Answer 24

Function: separate proteins or DNA fragments of different sizes.
Agarose gel covered with buffer solution. Electrodes at either end, DNA has -ve as many phosphate groups so moves towards positive electrode.
Smaller fragments move faster so travel further.

Question 25

What is the process of DNA profiling?

Answer 25

Extract DNA - quality increases with PCR
Digestion - restriction enzymes lead to smaller fragments
Comparison with banding patterns - separate by electrophoresis, banding patterns unique, compared to other individuals

Question 26

Compare DNA replication in vivo and in vitro.

Answer 26

Similarities: both need hydrogen bonds broken, single strand as template, DNA polymerase to form Phosphodiester bonds, DNA elongated 5’ to 3’
Differences: in vitro - heat at 95’, thermostable DNA polymerase, DNA primers, short strands only.
In vivo - DNA helicase, normal DNA polymerase, no primers, whole chromosomes.

Question 27

What are uses of electrophoresis?

Answer 27

Diagnose genetic conditions from mutated proteins
Identify inherited conditions
Locate specific gene for engineering
Presence of same gene for genome comparison studies

Question 28

How are proteins separated using electrophoresis?

Answer 28

Large tertiary structure may stop it moving from gel. R group depends if it has charge or not.
Heat to denature and expose charged R group. Add -ve detergent so all proteins have -ve so only separate by size/mass.

Transfer onto nylon membrane and view:
Visible stain, fluorescent labels and UV light, radioactive labels and x ray.

Question 29

Define DNA probe.

Answer 29

Short, single stranded pieces of DNA with base sequence complementary to known sequence.

Question 30

How are alleles located with DNA probes?

Answer 30

1. Digestion - restriction enzymes
2. Gel electrophoresis
3. Blotting - transfer to nylon membrane
4. Probe hybridisation - add probe, will bind to DNA if complementary sequence present
5. Washing - remove unbound probe
6. Detection - with x ray

Question 31

What are microarrays?

Answer 31

DNA probes are fixed to glass slide. DNA labelled fluorescently and complementary base pairing occurs and excess washed off.

Question 32

Define in vivo cloning.

Answer 32

Gene copies are made within a living organism. As grows and divides, replicates DNA to make multiple gene copies.
Can be used to make proteins eg. Insulin

Question 33

How is recombinant DNA made?

Answer 33

Use restriction endonuclease to cut gene out of DNA.
Cuts DNA at palindromic sequences to form sticky ends on isolated gene. Make sure has promoter and terminator.
Isolate vector’s DNA and cut it open with same restriction enzyme to ensure complementary sticky ends.
Mix gene with vector DNA and use DNA ligase to seal backbone.

Question 34

Describe the process for transforming a recipient cell.

Answer 34

Vector carrying recombinant DNA is used to transfer the gene.
- heat shock treatment - bacterial cells and alternating temps in CaCl2 to increase cell wall permeability and increase plasmid uptake.
- electroporation - high voltage to increase membrane permeability.
- electro function - electric currents
- transduction - DNA inserted into bacteriophage.

Question 35

How can transformed cells be identified?

Answer 35

Marker genes detect the presence of vector in cells.

Question 36

Define a transgenic organism.

Answer 36

Contains DNA from another organism.

Question 37

How is gene fragment obtained by reverse transcriptase?

Answer 37

The mRNA is a template for cDNA synthesis. Hydrolyse mRNA and make double stranded DNA with DNA polymerase and primers.
Introns already spliced out of gene - correctly transcribed and translated.

Question 38

How is a gene fragment obtained by restriction endonuclease?

Answer 38

Cut gene out of DNA by cutting backbone at specific palindromic sequences.
Forms blunt ends and sticky ends.

Question 39

Evaluate the use of GMOs.

Answer 39

Good: increase shelf life and crop yield and nutrient content of foods, aesthetics to consumers, resistant to external factors.
Bad: unknown long term affects on health, decreased gene pool and biodiversity, ethical issues, expensive.

Question 40

Define gene therapy.

Answer 40

Inserting a functional allele for a certain gene into a cell which only contains mutated/non functioning alleles.

Question 41

What are the different types of gene therapy?

Answer 41

Germ line - insert into gamete/zygote. All cells contain allele, inherited, not repeat procedure.
Somatic - insert into body cells, only some cells affected, not inherited, frequent repeats.

Question 42

How can an allele be delivered by a virus vector? And outline any issues with this.

Answer 42

Can be GM to contain functioning allele and not cause disease. Insert functioning allele into target cells.
Issues: virus may cause an immune response, immunity may develop. Little control where virus inserts gene into genome - middle of gene -> frameshift or into promoter region and affects on/off. Cell cycle and risk of tumours increases.

Question 43

How can an allele be delivered by liposomes? And outline issues with this.

Answer 43

Alleles are packaged into small spheres of lipid bilayer and passed through the plasma membrane.
Issues: allele not into nucleus, treatment repeated regularly, hard to reach target cells.