DNA Technology

Question 1

Name the stages involved in the process of making a protein using DNA technology.

Answer 1

Isolation - of DNA fragments with the gene for a desired protein
Insertion - of the DNA fragment into a vector
Transformation - transfer the DNA to a host cell
Identification - using gene markers
Growth/cloning - the host cells

Question 2

What is recombinant DNA?

Answer 2

The DNA of two different organisms that had been combined

Question 3

The use of reverse transcriptase

Answer 3

An enzyme that catalyses the production of DNA FROM RNA

Used to change the RNA from the retroviruses into DNA

Question 4

1. Isolation using reverse transcriptase

Answer 4

A cell that readily produces the protein is selected.
They have large quantities of the relevant mRNA
Reverse transcriptase is used to make DNA from RNA - cDNA (complimentary DNA)
DNA polymerase builds up the complimentary nucleotides on the cDNA template

Question 5

1. Isolation using restriction endonuclease

Answer 5

Enzymes that cut up viral DNA
Each one cuts a DNA strand at a specific sequence of bases - recognition sequence
Sometimes cut at two opposite base pairs (blunt ends)
Or a staggered fashion exposing unpaired bases (sticky ends)

Question 6

What does ‘in vivo’ mean?

Answer 6

The DNA fragments are cloned by transferred to a host cell using a vector

Question 7

What does ‘in vitro’ mean?

Answer 7

The DNA fragments are cloned using the polymerase chain reaction

Question 8

How is recombinant DNA formed?

Answer 8

The restriction endonuclease cuts out a DNA fragment, leaving a sticky end
DNA from another source cut with the same endonuclease joins the other section using DNA ligase

DNA ligase joins the phosphate-sugar framework

Question 9

What is a vector?

Answer 9

A carrying unit used to transport the DNA into the host cell

- usually a plasmid

Question 10

2. Insertion using restriction endonuclease

Answer 10

Restriction endonuclease breaks the plasmid loop at an antibiotic resistance gene
(The same restriction endonuclease used to cut out the DNA fragment)
DNA fragments are mixed with the opened up plasmid, becoming incorporated
Joined by DNA ligase
Recombinant DNA is formed

Question 11

3. Transformation

Answer 11

The plasmids are then reintroduced into bacterial cells.
Bacteria cells and the plasmids are mixed in a calcium ion medium, making the bacteria permeable for the plasmid to enter

Question 12

Why do not all the bacterial cells possess the DNA fragments?

Answer 12

Only a few bacterial cells take up the plasmids when they are mixed

Some plasmids do not incorporate the DNA fragment

Question 13

How are the antibiotic resistance genes used to identify which bacterial cells have taken up the plasmid?

Answer 13

Resistance to ampicillin and tetracycline

• All bacterial cells are grown in a medium containing ampicillin
• Bacteria cells that have taken up the plasmid have resistance to it
• The cells therefore survive
• The cells that don’t take it up aren’t resistant and die

Question 14

4. Identification using gene markers

Answer 14

Gene markers are:
Resistant to an antibiotic
Make a fluorescent protein
Produce an enzyme with identifiable actions

Question 15

Fluorescent markers

Answer 15

A gene from a jellyfish is transferred into the plasmid and produces a green fluorescent protein (GFP)
Those that have taken up the plasmid with the gene that is to be cloned will NOT produce GFP

Question 16

Enzyme markers

Answer 16

A gene that produces lactase as this turns a particular colourless substrate blue
If a plasmid with the required gene is present it will NOT produce lactase

Question 17

Antibiotic resistance markers

Answer 17

Replica plating - a technique to identify the cells with the plasmids that have taken up the new gene

This uses the other resistant gene in the plasmid - the one cut to incorporate the required gene
Gene for resistance to tetracycline is but so will no longer produce the enzyme to break down tetracycline

Question 18

Replica plating

Answer 18

The bacterial cells that survive the ampicillin have taken up the plasmid - these are cultures on an agar plate
A sample of each colony is transferred onto a replica plate
The replica plate contains tetracycline
Colonies that die took up the required gene
These can then be identified on the original plate

Question 19

PCR - what does this process require?

Answer 19

The DNA fragment to be copied 
DNA polymerase to join nucleotides 
Primers 
Nucleotides
Thermocycler - a computer-controlled machine that varies the temperature precisely

Question 20

What are primers?

Answer 20

Short sequences of nucleotides with complementary base pairs to those at one end of each of the two DNA fragments

Question 21

First stage of PCR

Answer 21

Separation of DNA stands
DNA fragments, primers and DNA polymerase are placed in the termocycler
Temperature increased to 95C causing the strands to separate

Question 22

Second stage of PCR

Answer 22

Addition (annealing) of primers
Mixture is cooled to 55C
Primers join to their complimentary bases on the DNA fragment
Provide the start sequence for DNA polymerase and prevents the two strands from rejoining

Question 23

Third stage of PCR

Answer 23

Synthesis of DNA
Temperature is increased to 72C - optimum temperature for DNA polymerase
Adds complimentary nucleotides along the DNA strands, beginning at the primer until it reaches the end of the chain

Question 24

Advantages of in vivo

Answer 24

Useful when introducing a gene into another organism
Almost no risk of contamination
Very accurate
Cuts out a specific gene so very precise
Can be produced in large quantities

Question 25

Advantages of in vitro

Answer 25

Extremely rapid - in a matter of hours 100 billion copies can be made Doesn't require living cells

Question 26

Use of recombinant DNA

Answer 26

``` Genetic modification - when the genetic make up of organisms are altered by transferring genes they can: Increase yield of animals or crops Increase nutrient content of food Introduce resistance to disease or pests Make plants tolerant to herbicides Develop tolerance to extreme weather ```

Question 27

Genetically modified microorganisms

Answer 27

Antibiotics - naturally produced by bacteria GM can increase quantity of antibiotics produced Hormones- insulin can be incorporated into bacterial cells with no effects to the patient Enzymes - manufactured by modified bacteria

Question 28

What causes cystic fibrosis?

Answer 28

A mutant recessive allele where there's a deletion mutation CFTR gene produces a non functional protein CFTR is a chloride- ion channel protein

Question 29

What happens in a person with cystic fibrosis?

Answer 29

The non functional channel protein prevents chloride ions from leaving the cell Water is retained in the cell as the concentration gradient causes osmosis into the cell Causes breathing difficulties Thick mucus in the pancreatic ducts and sperm ducts Mucus congestion leading to risk of infection

Question 30

Treating cystic fibrosis using gene therapy: gene replacement

Answer 30

The defective gene is replaced with a healthy gene. Germ-line gene therapy - replacing the gene in the fertilised egg All cells will develop normally and be passed on to their offspring Permanent solution affects future generations Process is illegal due to ethical issues

Question 31

Treating cystic fibrosis using gene therapy: gene supplementation

Answer 31

Copies of the healthy gene are added alongside the defective, masking the defective gene Somatic-cell gene therapy - targets affected tissues Not passed onto offspring The cells continually die and are replaced so treatment is needed periodically- limited success Long term is to target undifferentiated stem cells therefore lasting their lifespan

Question 32

How is the cloned CFTR genes delivered in somatic-cell gene therapy? A harmless virus

Answer 32

Adenovirus causes colds by injecting their DNA into the epithelial cells of the lungs Useful vectors to deliver this gene then In vivo gene cloning is used The adenoviruses are introduced it or he nostrils of the patient

Question 33

How is the cloned CFTR genes delivered in somatic-cell gene therapy? Lipid molecules

Answer 33

Genes are wrapped in lipid molecules as these can easily pass through the phospholipid bilayer In vivo gene cloning is used The plasmids are removed from the bacteria and wrapped in lipid molecules forming liposomes These are sprayed into the nostrils of the patient and drawn down into the lungs

Question 34

Problems with how CFTR is delivered in gene therapy

Answer 34

Adenoviruses may cause infections Patients may develop immunity to the adenoviruses Liposomes may be too large to pass through May not be expressed

Question 35

What is Severe combined immunodeficiency (SCID)?

Answer 35

A rare inherited disorder where the sufferer doesn't show a cell mediated immune response or produce antibodies Arises when the individual inherits a defect in the gene that codes for the enzyme ADA ADA destroys toxins that would kill WBC

Question 36

How is SCID treated?

Answer 36

ADA from a healthy human is inserted into a retrovirus They're mixed with the patients T cells ADA is inserted into the T cells T cells are reintroduced into the patients blood Success is limited because T cells only live for 6-12 months so has to be repeated

Question 37

What is a DNA probe?

Answer 37

A short, single-stranded section of DNA that has a label attached that makes it easily identifiable Two common ones used are: Radioactively labelled probes Fluorescently labelled probes

Question 38

How are DNA probes used?

Answer 38

Has complimentary bases to a portion of DNA that makes up part of the gene whose position we want to find DNA being tested has been treated to separate its two strands Separated DNA strands are mixed with the probe which binds to complimentary bases on the strand - DNA hybridisation Site at which it binds can be identified

Question 39

DNA sequencing | What is the Sanger method?

Answer 39

Uses modified nucleotides that cannot attach to the next base sequence when they are being joined together They act as terminators, ending synthesis of a DNA strand

Question 40

What is in the four test tubes set up for the Sanger method?

Answer 40

Single stranded DNA fragments which act as a template for the synthesis of it's complimentary strand Mixture of nucleotides Small quantity of one of the 4 terminator nucleotides (tube 1 - Adenine etc) A primer to start DNA synthesis (labelled) DNA polymerase to catalyse DNA synthesis

Question 41

Why are there different lengths of DNA fragments in DNA sequencing?

Answer 41

Binding of nucleotides is a random process so the chance of either a terminator or normal nucleotide binding is equally likely DNA synthesis may be terminated after a few nucleotides or after a long fragment, depending on where the terminator binds

Question 42

How can the section of DNA be identified in DNA sequencing?

Answer 42

DNA fragments will all end in the same base (tube 1 - Adenine etc) They can be identified because the primer attached to the other end of the DNA section is labelled

Question 43

What is the stage in which the different lengths of DNA fragments are separated out?

Answer 43

Gel electrophoresis

Question 44

What happens in gel electrophoresis?

Answer 44

The DNA fragments are placed on an agar gel A voltage is applied across it Larger fragments move slower so smaller ones move further A sheet of photographic film is placed over the agar gel Radioactivity from each fragments exposes the film showing where it is situated on the gel

Question 45

DNA fragments sequenced have to be less than around 500 bases.

Answer 45

Larger genes and whole genomes must therefore be cut into smaller fragments by RESTRICTION ENDONUCLEASE

Question 46

What does restriction mapping involve?

Answer 46

Involves cutting DNA with a series of different restriction endonucleases.

Question 47

How are the fragments separated in restriction mapping?

Answer 47

By electrophoresis

Question 48

How is DNA sequencing and restriction mapping carried out by machines?

Answer 48

A fluorescent dye is used as a label the DNA primer instead of a radioactive labels Each base takes up a different colour Electrophoresis is carried out and results are scanned by lasers to show what the bases are