3.4.10 Recombinant DNA Technology

Question 1

What is recombinant DNA?

Answer 1

DNA from 2 different sources are combined

Question 2

What does the production of DNA fragments involve?

Answer 2

Transferring a fragment of DNA from one organism to another

Question 3

Explain why the recipient and donor organisms don’t have to be same species when transferring a fragment of DNA from one organism to another

Answer 3

• ∵ genetic code is universal
  
  • (same DNA base triplets code for same amino acids in all living things)
• ∵ transcription and translation mechanisms are pretty similar

Question 4

Name 3 methods of making DNA fragments

Answer 4

• Using Reverse Transcriptase
• Using Restriction Endonuclease Enzymes
• Using a ‘Gene Machine’

Question 5

Why is mRNA easier to obtain than DNA?

Answer 5

Many mRNA molecules (complementary to a gene)

Question 6

What does reverse transcriptase do?

Answer 6

Makes DNA from an RNA template

Question 7

What is the DNA produced by reverse transcriptase called?

Answer 7

complementary DNA (cDNA)

Question 8

Describe how you can make DNA fragments using reverse transcriptase

Answer 8

1. mRNA is isolated from cells
2. Its mixed with free DNA nucleotides and reverse transcriptase
3. Reverse transcriptase uses mRNA as a template to synthesis a new strand of cDNA
4. DNA polymerase is used to build up the complementary base pairings with cDNA = DNA is formed

Question 9

Some sections of DNA have ________ sequences of nucleotides

Answer 9

palindromic

Question 10

What are palindromic sequences of nucleotides?

Answer 10

Sequences that consist of antiparallel base pairs

(Base pairs that read the same in opposite directions)

Question 11

What are restriction endonucleases?

Answer 11

Enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) DNA at these places

Question 12

Why do different restriction endonucleases cut at different specific recognition sequences?

Answer 12

∵ shape of recognition sequence is complementary to enzyme’s active site

Question 13

When can you use restriction endonucleases to create a DNA fragment?

Answer 13

If recognition sequences are present either side of DNA fragment

Question 14

Describe how you can use restriction endonuclease to produce a DNA fragment

Answer 14

1. DNA sample is incubated with specific restriction endonuclease & it cuts DNA fragment out via hydrolysis reaction
2. Sometimes cut leaves sticky ends - small tails of unpaired bases at each end of the fragment
3. Sticky ends can be used to bind (anneal) DNA fragment to another pieces of DNA that has sticky ends with complementary sequences

Question 15

What is meant by a ‘gene machine’?

Answer 15

• Technology that enables fragments of DNA that can be synthesised from scratch without need for pre-existing DNA template
  
  • Database contains necessary info to produce DNA fragments

Question 16

What does a ‘gene machine’ allow you to do?

Answer 16

Can produce DNA sequences that don’t exist naturally

Question 17

Describe how you can use a ‘gene machine’ to produce a DNA fragment

Answer 17

1. Sequence required is designed
2. 1st nucleotide in sequence is fixed to some sort of support
   
   • e.g. a bead
3. Nucleotides are added step by step in correct order, in a cycle of processes that include adding protecting groups
4. Short sections of DNA (called oligonucleotides - 20 nucleotides long) are produced
5. Once complete, they’re broken off from support and all protecting groups are removed
6. Oligonucleotides can then be joined together to make longer DNA fragments

Question 18

Using a ‘Gene Machine’ to Producing DNA Fragments

Explain why protecting groups are added when nucleotides are added step by step in correct order

Answer 18

Protecting groups make sure nucleotides are joined at the right points, to prevent unwanted branching

Question 19

Name 2 methods of amplifying (makes lots of copies of) DNA fragments

Answer 19

• In Vivo Amplification
• In Vitro Amplification

Question 20

Name the 3 stages in in vivo amplification

Answer 20

1. DNA Fragment is Inserted into Vector
2. Vector Transfers DNA Fragment into Host Cells
3. Identifying Transformed Host Cells

Question 21

In Vivo Amplification

What vectors are normally used?

Answer 21

Can be plasmids or bacteriophages (viruses that infect bacteria)

Question 22

What is a recombinant plasmid?

Answer 22

When a gene is added from another organism to the plasmid

Question 23

In Vivo Amplification

1) Describe how a DNA fragment is inserted into a vector

Answer 23

1. Vector DNA is cut open using same restriction endonuclease that was used to isolate DNA fragments containing the target gene
   
   • So sticky ends of vector are complementary to sticky ends of DNA fragment containing the gene
2. Vector DNA and DNA fragment are mixed together with DNA ligase
   
   • DNA ligase joins sticky ends of DNA fragment to sticky ends of vector DNA
     
     • aka ligation

Question 24

Describe how a plasmid vector transfers recombinant DNA into host cells

Answer 24

Host cells are to be persuaded to take in the plasmid vector and its DNA

Question 25

Describe how a bacteriophage vector transfers recombinant DNA into host cells

Answer 25

* Bacteriophage infect host bacterium by injecting its DNA into it * Phage DNA (with the target gene in it) then integrates into bacterial DNA

Question 26

Host cells that take up vectors containing the gene of interest are said to be \_\_\_\_\_\_

Answer 26

transformed

Question 27

What are used to identify transformed cells?

Answer 27

Marker genes

Question 28

When are marker genes inserted into vectors and why?

Answer 28

* Inserted at same time as target gene (i.e. gene to be cloned) * ∴ transformed host cells will contain target gene and marker gene

Question 29

What are host cells grown on?

Answer 29

Agar plates Each cell divides and replicates its DNA, creating colony of cloned cells

Question 30

Name 3 examples of marker genes

Answer 30

* Genes that code for antibiotic resistance * Genes that code for fluorescence * Enzyme markers

Question 31

Describe how genes that can code for antibiotic resistance can be used as marker genes

Answer 31

Host cells are grown on agar plates containing specific antibiotic so only transformed cells with marker genes will grow

Question 32

Describe how genes that can code for fluorescence can be used as marker genes

Answer 32

Under UV light only transformed cells will fluoresce

Question 33

Describe how enzyme markers can be used as marker genes

Answer 33

Gene produces lactase and lactase will turn colourless substate blue

Question 34

Describe replica plating

Answer 34

1. Bacteria are placed into a medium containing e.g. ampicillin, with all bacteria that have taken up the plasmid (with gene for resistance to ampicillin) surviving 2. Each separate cell then grows into a genetically identical colony 3. A tiny sample of each colony is then placed into a medium containing tetracycline 4. The cells that are destroyed contain the recombinant DNA * Shows new gene has been taken up as gene for resistance to tetracycline is made useless

Question 35

What does a vector need to contain, if you want transformed host cells to produce a protein coded by DNA fragment?

Answer 35

Vector contains specific promoter and terminator regions

Question 36

**Producing Proteins requires Promotor and Terminator Regions** What happens if the vector doesn't contain the right promoter region?

Answer 36

DNA fragment won’t be transcribed by host cell and protein won’t be made

Question 37

What do promoter regions do?

Answer 37

DNA sequences that tell enzyme RNA polymerase when to start producing mRNA (Terminator regions tell it when to stop)

Question 38

**Producing Proteins requires Promotor and Terminator Regions** Promoter and terminator regions may be present in the ____ \_\_\_\_ or may be added in along with the \_\_\_\_\_

Answer 38

Promoter and terminator regions may be present in the _vector_ _DNA_ or may be added in along with the _fragment_

Question 39

How are copies of DNA fragments in *in vitro* made?

Answer 39

Use polymerase chain reaction (PCR) (copies of DNA fragments are made outside of living organisms)

Question 40

What is the benefit of using PCR?

Answer 40

It can be used to make millions of copies of a fragment of DNA in a few hours

Question 41

Describe how DNA fragments are made in *in vitro* amplification

Answer 41

1. Reaction mixture set up containing DNA sample, free nucleotides, primers and DNA polymerase * DNA polymerase creates new DNA strands 2. DNA mixture is heated to 95°C to break hydrogen bonds between 2 strands of DNA * Strands separate 3. Mixture is cooled to between 50°C and 65°C so primer can bind (anneal) to strands 4. Nucleotides attach by complementary base pairing 5. Reaction mixture is heated to 72°C so DNA polymerase can work 6. DNA polymerase join free DNA nucleotides alongside each template strand 7. 2 new copies of fragment of DNA formed & 1 cycle of PCR is complete 8. Cycle starts again, with mixture heated to 95°C and all 4 strands (2 original and 2 new) are used as templates 9. Each PCR cycle doubles the amount of DNA

Question 42

***In Vitro* Amplification** In the 3^rd PCR cycle, how many DNA fragments are produced?

Answer 42

16 DNA fragments * 1^st cycle = 2 x 2 = 4 DNA fragments * 2^nd cycle = 4 x 2 = 8 DNA fragments * 3^rd cycle = 8 x 2 = 16 DNA fragments

Question 43

How are transformed organisms produced?

Answer 43

Through genetic engineering

Question 44

Describe how transformed microorganisms can be produced

Answer 44

* Can be made using same technology as in vivo cloning * e.g. foreign DNA inserted into microorganisms to produce lots of useful protein

Question 45

Describe how transformed plants can be produced

Answer 45

* Gene codes for desirable protein inserted into plasmid * Plasmid is added to bacterium and bacterium is used as vector to get gene into plant cells * If right promoter region has been added along with gene, transformed cells will be able to produce the desired protein

Question 46

Describe how transformed animals can be produced

Answer 46

* Gene codes for desirable protein can be inserted into early animal embryo or into egg cells of a female * If gene inserted into a very early embryo, all body cells of resulting transformed animal will contain the gene * Inserting into egg cells = when female reproduces = all cells of offspring contain the gene

Question 47

Promoter regions that are only activated in specific cell types can be used to control...

Answer 47

exactly which of animal's body cells the protein is produced in

Question 48

What is the benefit of producing a protein in certain cells?

Answer 48

It can be harvested more easily

Question 49

What happens when a protein is produced in the wrong cells?

Answer 49

Damages the organism

Question 50

State some agricultural benefits of using recombinant DNA technology

Answer 50

1. Agriculture crops can be transformed to they give higher yields or are more nutritious * Means plants can be used to reduce risk of famine and malnutrition 2. Crops can be transformed to have pest resistance = fewer pesticides are needed * Reduces costs and reduces any environmental problems associated with using pesticides

Question 51

State some industrial benefits of using recombinant DNA technology

Answer 51

* Industrial processes often use enzymes * Enzymes can be produced from transformed organisms = produced in large quantities for less money = reducing costs

Question 52

State some medical benefits of using recombinant DNA technology

Answer 52

1. Many drugs and vaccines are produced by transformed organisms, using recombinant DNA technology * Can made quickly, cheaply and in large quantities using this method * So more people can afford them 2. Recombinant DNA technology has the potential to be used in gene therapy to treat human diseases

Question 53

What does gene therapy involve?

Answer 53

Altering the defective genes (mutated alleles) inside cells to treat genetic disorders and cancer

Question 54

State some agricultural disadvantages of using recombinant DNA technology

Answer 54

1. Farmers might plant only 1 type of transformed crop (monoculture) 1. Makes whole crop vulnerable to same disease ∵ plants are genetically identical 2. Monoculture also reduces biodiversity = damages the environment

Question 55

State some industrial disadvantages of using recombinant DNA technology

Answer 55

1. Anti-globalisation activists oppose globalisation 1. A few, large biotechnology companies control some forms of genetic engineering 2. As use of this technology increases = companies get bigger and more powerful 3. May force smaller companies out of business 2. Without proper labelling, some people think they won't have a choice about whether to consume food made using genetically engineered organisms 3. Some consumer markets (e.g EU) won't import GM food and products 1. Can cause economic loss to producers who have traditionally sold to those markets

Question 56

State some medical disadvantages of using recombinant DNA technology

Answer 56

1. Companies who own genetic engineering technologies may limit the use of technologies that could be saving lives 2. Some people worry this technology could be use unethically 1. e.g. make designer babies = illegal

Question 57

What is the role of a primer in PCR?

Answer 57

Enables replication/sequencing to start

Question 58

What is are DNA primers?

Answer 58

Short lengths of single-stranded DNA that are complementary to a particular sequence of DNA