3.8.4.1 Recombinant DNA Technology (Unit 8 Gene Tech)

Question 1

What is meant by the term ‘recombinant DNA technology’?

Answer 1

transferring DNA fragments from one organism or species to another.

Question 2

Why does recombinant DNA technology work so well?

Answer 2

the genetic code is universal as are the processes transcription and translation mechanisms.

Question 3

What does universal mean?

Answer 3

all organisms have the same amino acid coded for by the same DNA codon (triplet code).

Question 4

How are DNA fragments obtained?

Answer 4

• conversion of mRNA to DNA (cDNA) using reverse transcriptase.
• cut out of DNA using restriction enzymes.
• creating a new gene in a ‘gene macnine’.

Question 5

Via which two methods can DNA be amplified?

Answer 5

in vivo and in vitro

Question 6

What does in vivo mean?

Answer 6

carried out inside a living body

Question 7

What does in vitro mean?

Answer 7

carried out outside of a living body e.g. in a test tube

Question 8

What does PCR stand for?

Answer 8

polymerase chain reaction

Question 9

Is PCR in vivo or in vitro?

Answer 9

in vitro

Question 10

When obtaining DNA fragments using restriction enzymes what must be added to the start and end of the desired gene?

Answer 10

promoter and terminator regions

Question 11

When obtaining DNA fragments using restriction enzymes what is the name of the item that transports the desired gene into the host cell?

Answer 11

vector (plasmids for bacterial host cells)

Question 12

When obtaining DNA fragments using restriction enzymes what enzyme must be used to bind the phosphate-sugar framework of the desired gene into the DNA (preferable with ‘sticky ends’)?

Answer 12

DNA ligase

Question 13

What is the name given to DNA which consists of genetic material from two different organisms?

Answer 13

recombinant DNA

Question 14

What is the name given to an organism which contains genetic material from two different organisms?

Answer 14

GMO (genetically modified organism) and transgenic as it has genetic information from more than one sources.

Question 15

What are the stages involved in making a GMO?

Answer 15

1. isolation
2. insertion
3. transformation
4. identification
5. growth / cloning

Question 16

When making a GMO explain what happens in stage 1. (isolation)

Answer 16

The gene is isolated using 1 of 2 methods:

1. reverse transcriptase is used to obtain DNA from mRNA
2. restriction endonuclease enzymes are used to cut the desired gene from the DNA

Question 17

When making a GMO explain what happens in stage 2. (insertion)

Answer 17

RE’s cut at specific recognition sites to make either blunt or sticky ends. Sticky ends are better as there are bases which will H bond the bases together, as well as the phosphate-sugar connection (made by DNA ligase enzyme). Promoter and terminator bases sequences are also added to initiate start and end of the gene being transcribed. The desired gene is then inserted into a vector (most commonly a plasmid) which has had its DNA cut with the same RE so that there is a palindrome.

Question 18

When making a GMO explain what happens in stage 3. (transformation)

Answer 18

once the desired gene has been successfully incorporated into the vector it must be reintroduced to the host cell (e.g. plasmid into bacteria). Calcium ions and temperature changes are used to make the membrane permeable to allow the vector to pass into the cell.

Question 19

Why are all of the vectors not successfully taken up by the host cell?

Answer 19

• only some bacterial cells take up the recombination DNA (e.g bacterial cells taking up the plasmid).
• some vectors (e.g plasmids) have not taken up the desired gene.
• sometimes multiple fragments join together to form their own plsmids

Question 20

When making a GMO explain what happens in stage 4. (isolation)

Answer 20

marker genes are used to identify which vector has successfully taken up the desired gene, and which host cell has successfully taken up the vector. Markers used are anti-biotic resistance, enzyme and fluorescence.

Question 21

How does stage 4 (identification) work with the anti-biotic resistance method?

Answer 21

replica plating:

• The cells that survived the first antibiotic have taken up the plasmid.
• Those cells are cultured by spreading them thinly on nutrient agar plates.
• Each cell on the plate will become a culture of clones.
• A small sample from each colony is transferred to another plats (the replica plate) and placed in the same position as on the original plate.
• The replica plate contains the second antibiotic against which the bacterial cells will have no resistance if they have the plasmid with the DNA fragment (as the resistance gene will have been removed).
• Colonies killed on the replica plate are the ones with the required gene.
• The same ones on the original plate are alive and can be cultures further.

Question 22

How does stage 4 (identification) work with the enzyme method?

Answer 22

• the enzyme gene marker codes for the production of the protein enzyme lactase.
• Lactase turns a certain substance from clear to blue.
• the desired gene is placed in the centre of the lactase coding gene.
• If the plasmid with the required gene is present in the bacterial cells the colony grown from it will not produce lactase and therefore not cause the colour change to blue whilst the undesired cells will cause a colour change.

Question 23

How does stage 4 (identification) work with the fluorescence method?

Answer 23

• A more recent and faster method is the transference from a jellyfish plasmid which produces a green fluorescent protein (GFP).
• The desired gene to be cloned is transferred to the centre of the GFP gene so any bacterial cells which have taken up the desired gene will not express the GFP so they will not fluoresce whilst the other cells without the desired gene will.
• There is no need for replica plating as the desired cells have not been harmed. All is required is to retain the cells which do not fluoresce by viewing them under a microscope.

Question 24

Where is the reverse transcriptase used in obtaining a DNA fragment (stage 1. isolation) come from?

Answer 24

Retrovisuses are human viruses of which human immunodeficiency virus (HIV) is the best known.
They have RNA as their genetic information but they can synthesise DNA from the RNA using REVERSE TRANSCRIPTASE (RT) (an enzyme).

Question 25

How does using reverse transcriptase to isolate a single gene work?

Answer 25

• A cell that produced the protein is selected.
  (e. g. Beta-cells of the islets of Langerhans in the pancreas produce insulin)
• The mRNA is extracted from the cells.
• RT is used to make DNA from RNA. This is known as COMPLIMENTARY DNA (cDNA) as the DNA bases are complimentary to the RNA bases.
• To make the other strand of DNA the enzyme DNA POLYMERASE is used to build up the complimentary nucleotides on the cDNA template.
• The resulting double strand of DNA is the required gene!!!

Question 26

Why do bacterial cells have restriction endonuclease enzymes naturally?

Answer 26

Bacteria are often invaded by viruses (bacteriophages) which inject foreign DNA into their cells and so the bacteria have enzymes which cut up the viral DNA. These are RESTRICTION ENDONUCLEASES (RE).
There are many different RE’s. Each cuts the DNA at a different sequence of bases called the recognition sequence.

Question 27

What is another name for the recognition site that the restriction endonuclease cut at?

Answer 27

cleavage site

Question 28

Once all of the stages for producing a GMO with recombinant DNA have been completed, there is only one thing left to do. Grow or clone the new organisms. If your GMO is a bacteria what must you do to ensure maximum growth?

Answer 28

Ensure the conditions for bacterial growth are optimum (e.g. suitable temperature, water availability, Oxygen and a nutrient source).

Question 29

What is ‘nick name’ for the machine used to create genes in the laboratory?

Answer 29

the gene machine

Question 30

Before we started to use gene technology to create new organisms, how did breeders and farmers alter their livestock?

Answer 30

selective breeding over many generations.

Question 31

Give some uses of DNA technology

Answer 31

examples:
Increased yield of farm animals and crops.
Improved nutrient content of food.
Introducing disease and pest resistance to animals and plants.
Making crop plants herbicide tolerant.
Developing tolerance to environmental conditions.
Making vaccines.
Producing medicines and treatment of disease.

Question 32

What items/apparatus are needed to perform PCR?

Answer 32

• The DNA fragment to be copied.
• DNA polymerase to join together the nucleotides in a hot environment.
• Primers with short, set nucleotides complementary to those on the ends of the fragment to join to the ends of the DNA fragment. 15-20 bases long.
• Nucleotides of all ATC and G to bind to complementary bases.
• Thermocycler to control temperature changes during PCR.

Question 33

What temperature is needed to separate the DNA strands in PCR?

Answer 33

Heat the contents of the thermocycler to 95°C to separate the DNA strands.

Question 34

What temperature is needed to for primers to anneal in PCR?

Answer 34

Cool the thermocycler contents to 55°C. This causes the annealing primers to attach (anneal) to the start and end complementary bases on the DNA strands. (this also keeps the strands apart).

Question 35

What temperature is needed for DNA to bind new nucleotides in PCR?

Answer 35

Heat up to 72°C. This is the optimum temperature for DNA polymerase to cause new nucleotides to bind to their complementary base pairs.

Question 36

Why do we use PCR?

Answer 36

PCR is the basis of Genetic Fingerprinting, where a sample form a known source can be compared against a sample.
It allows even a tiny sample of skin, blood, a hair follicle or semen to contain enough DNA to be amplified in PCR.

Question 37

Why is a thermocycler used in PCR?

Answer 37

PCR uses a computer controlled machine which allows temperatures to be controlled and timed accurately,
The whole temperature cycle takes around 2 minutes so over 1 million copies of the DNA can be made in 25 cycles, and 100 billion can be made in a few hours.

Question 38

Where do we get thermostable enzymes from to use in PCR?

Answer 38

thermostable enzymes are from thermophilic bacteria (e.g. Thermus aquaticus) which survive in hot springs which join nucleotides together.

Question 39

What are the advantages of in vivo coning?

Answer 39

• Can add a gene to another organism via vectors and plasmids.
• Low risk of contamination as the same RE is used to cut the same codes for all DNA involved so no stray DNA is present.
• Only desired genes are cut out.
• Produces transformed bacteria to produce many products (e.g. insulin).
• No prior knowledge of bases sequences is needed.

Question 40

What are the advantages of in vitro coning?

Answer 40

• Quick. Can make billions of copies from a small sample in a few hours.
• No living cells are needed.

Question 41

What are the disadvantages of in vitro coning?

Answer 41

• Cannot add genes to other organisms.
• A very pure sample is needed as contaminants can also be multiplied giving false results.
• The whole DNA fragments are copied.
• Only magnifies DNA sample.
• Prior knowledge is needed to synthesise primers.

Question 42

What are the advantages of in vivo coning?

Answer 42

• Takes days or weeks to make billions of copies from a small sample.
• Requires living cells and growing cultures.

Question 43

What are primers and how are they used in PCR?

Answer 43

Short pieces of DNA with set bases complementary to those on DNA sample. They attach to the start and end of the DNA to provide start and stop instructions for PCR. DNA polymerase can only attach nucleotides at the end of an existing chain. Primers also keep the 2 strands apart.

Question 44

Why are 2 different primers needed in PCR?

Answer 44

The sequences at the 2 ends of the strand are different

Question 45

What bond is broken when DNA strands are separated in PCR?

Answer 45

H Bonds

Question 46

What could happen if contaminant biological material exists in the sample?

Answer 46

Any contaminant would also be copied.

Question 47

role of DNA/Taq polymerase

Answer 47

joins nucleotides together in DNA (complementary strand)

Question 48

name 2 enzymes involved in inserting DNA fragments into plasmids

Answer 48

ligase

restriction endonucleases

Question 49

describe the role of ligase

Answer 49

joins gene/DNA into the plasmid/vector

Question 50

describe the role of restriction endonuclease when used to add a piece of DNA into a plasmid

Answer 50

cuts the plasmid

Question 51

What is reverse transcriptase

Answer 51

Enzyme from HIV, it converts RNA into DNA

Question 52

How can reverse transcriptase be used in genetic engineering?

Answer 52

Mature mRNA is extracted and converted to cDNA by Reverse Transcriptase
cDNA is converted to double stranded DNA by DNA polymerase

Question 53

What is an advantage of using reverse transcriptase in genetic engineering?

Answer 53

mRNA is found in large amounts in cells

mRNA contains no introns, so the gene products are easily expressed even by bacteria

Question 54

What is a restriction endonuclease?

Answer 54

DNA is cut at specific recognition sites

Cuts can form ‘sticky ends’ these are complementary and can be used for joining DNA together

Question 55

How is a gene machine used?

Answer 55

Desired gene sequence is designed using a computer – it is checked for safety and standards.
Small pieces of DNA (oligonucleotides) are synthesised and joined together to make a sequence of DNA
Using sticky ends these pieces of DNA can be inserted into a vector that can be used in cloning.

Question 56

What is the advantage of using a gene machine?

Answer 56

Gene machines are quicker as they don’t need to isolate DNA/mRNA first

Question 57

Describe the whole model answer for genetic engineering

Answer 57

Isolate DNA/mRNA from an organism using a restriction endonuclease or reverse transcriptase to get DNA
Cut plasmid and isolated DNA with the same restriction endonuclease to get complementary sticky ends
A promoter and terminator region may need to be added to the gene
Use DNA ligase to join the desired gene to a plasmid – forming phosphodiester bonds
Include marker gene e.g. antibiotic resistance
Transform host using Ca2+ ions and a heat shock so plasmid passes through the membrane
Allow bacteria to grow (colonies) then (replica) plate onto medium where the marker gene is expressed
Bacteria / colonies not killed have antibiotic resistance gene/don’t fluoresce/have enzyme action and (probably) the wanted gene;

Question 58

Describe the whole PCR cycle

Answer 58

DNA heated to 90 to 95°C to separate the strands
DNA is cooled to 55°C so that primers can bind
Free nucleotides attach by complementary base pairing
Temperature is increased to 72°C and DNA polymerase joins nucleotides together forming a phosphodiester bond
Cycle is repeated and DNA is copied at an exponential rate

Question 59

Why will the PCR reaction eventually plateau/stop

Answer 59

The cycle will eventually stop when the nucleotides and primers run out.

Question 60

Why is Taq polymerase used in PCR

Answer 60

Taq polymerase is thermostable, it works at high temperatures and doesn’t denature.

Question 61

What are the applications of PCR

Answer 61

PCR can be used in Crime scene analysis and genotyping (and lots more)

Question 62

Describe Gel Electrophoresis

Answer 62

DNA is cut at areas of tandem repeats using restriction endonucleases
DNA fragments are placed in wells at the top of an agar gel.
An electric current is applied over it.
DNA is negatively charged due to the phosphate group
The DNA moves towards the positive electrode, but at different rates.
Small fragments move further through the gel
A ladder/marker can be used to determine the size of the DNA fragments

Question 63

Describe the process of DNA fingerprinting

Answer 63

Extracted DNA is cut with a restriction endonuclease at sites of variable number tandem repeats/minisatellites
DNA is separated by gel electrophoresis, shorter fragments run further on the gel
Use Southern Blotting to transfer DNA to a nylon membrane
Use an alkaline solution to make DNA single stranded
Add a single stranded probe tagged with radioactive/fluorescent molecule
Visualise the DNA using and X-ray film or UV light

Question 64

What cells in an animal should you genetically modify?

Answer 64

Early-stage embryos, as the gene will be present in most cells in the organism
Allows all cells to make the protein

Question 65

Where do you insert the gene and why?

Answer 65

Insert a promoter into genes that are produced and easily extracted e.g. milk
Extracting proteins from other places may harm the organism
Expressing proteins in all cells may cause harm