Section 8 - The control of gene expression: 21. Recombinant DNA technology

Question 1

What is Recombinant DNA

Answer 1

DNA of two different organisms combined together
- Resulting organism is a ‘transgenic’ or ‘Genetically modified’ (GM) organism
- Possible due to the universal nature of the genetic code
- Transcription and translation are universal, so recombinant DNA can lead to the production of proteins in GM organisms
eg. Production of insulin without the use of donor tissue

Question 2

What are the 3 ways of producing DNA fragments for use in Recombinant DNA technology

Answer 2

• Reverse transcriptase
• Restriction endonuclease
• Gene Machine

Question 3

What is reverse transcriptase

Answer 3

Enzyme that catalyses the production of DNA from RNA
- Used by retroviruses, such as HIV, as their genetic code is stored as RNA, used to produce DNA
- Can be used to produce the required DNA fragments for recombinant DNA technology, from the relevant RNA in a host cell

Question 4

How is reverse transcriptase used to produce DNA fragments required for Recombinant DNA technology

Answer 4

• A cell that readily produces the required protein is selected (eg. β-Cells that produce insulin)
• Relevant mRNA is extracted
• ‘Reverse transcriptase’ is then used to make DNA from this RNA, made of complementary nucleotides
• Produces ‘complementary DNA’ (cDNA)
• To make the required strand, the enzyme ‘DNA polymerase’ is used (using cDNA as a template)
• ∴ Required gene is then released as double stranded DNA

Question 5

What are restriction endonucleases

Answer 5

Enzymes that can cut up sections of DNA
- ‘Break’ phosphodiester bonds between nucleotides in the sugar-phosphate backbone
- ‘Break’ hydrogen bonds between the two strand of the double helix

Question 6

How are restriction endonucleases used to produce DNA fragments required for Recombinant DNA technology

Answer 6

• Required section of DNA is identified within a base sequence
• A Restriction endonuclease enzyme is used to cut out this fragment (cut at recognition sites)
• This releases the required DNA fragment, with overhanging bases on each end (sticky ends)

Question 7

What are ‘sticky ends’ and why are they important when forming recombinant DNA

Answer 7

When cut with restriction endonucleases, the end of the DNA sequence is staggered between the strands, with overhanging bases (sticky ends)
- If the same restriction endonuclease is used to cut DNA, all produced fragments will have complementary sticky ends
- ∴ Overhanging strands can join together through the use of the enzyme ‘DNA ligase’
- This allows the DNA of one organism to be combined with that of another (recombinant DNA)

Question 8

How is the gene machine used to produce DNA fragments required for Recombinant DNA technology

Answer 8

• The required sequence of nucleotide bases is determined from the amino acid sequence of the desired protein
• Sequence is checked for biosafety, biosecurity and to make sure it meets ethical requirements
• The computer designs a series of small, overlapping, single strands of nucleotides called ‘oligonucleotides’, which can be assembled into the desired gene
• The ‘oligonucleotides’ are joined together, with the complementary strand produced in PCR to give required DNA section
• No introns, or non-coding sections, so able to be used to produce the required protein

Question 9

What are the advantages of using the gene machine to produce DNA fragments required for Recombinant DNA technology

Answer 9

• Any base sequence can be produced quicky
• High level of accuracy
• No introns or non-coding DNA sections
  ∴ Can be transcribed and translated by prokaryotic cells (bacterial host) to produce the required protein

Question 10

What is ‘In Vivo’ cloning

Answer 10

The process by which required DNA fragments are cloned through the use of vectors and bacterial hosts

Question 11

How are DNA fragments prepared before ‘In Vivo’ cloning can take place

Answer 11

As the cloned recombinant DNA will later be used to produce proteins, 2 extra lengths of DNA are added to the fragments, to allow transcription to occur
- Promoter:
- Length of nucleotide bases that transcriptional factors and RNA polymerase will bind to
- ∴ Allows transcription to occur
- Terminator:
- Length of DNA that causes RNA polymerase to be released, ending transcription at the required point
- Ensures that only the required protein is produced by the recombinant DNA

Question 12

What are the main stages of ‘In Vivo’ cloning

Answer 12

• Insertion of DNA fragments into a Vector (bacterial plasmid)
• Introduction of the DNA into host for replication (transformation)
• Identification of required DNA with marker genes

Question 13

What is a vector in the process of ‘In Vivo’ cloning

Answer 13

Carrying unit, used to transport the DNA into a host cell for replication (bacterial plasmid)

Question 14

What is the process of inserting DNA fragments into a vector for ‘In Vivo’ cloning

Answer 14

• Once the required DNA fragment has been isolated and has the correct promoter/terminator regions, it is ready to be added to a vector
• Bacterial plasmids with a specific marker gene are chosen, so the recombinant plasmids can be identified later
• The same restriction endonuclease is used to cut the fragments and the plasmids, resulting in complementary sticky ends
• DNA fragments are then incubated with the cut plasmids and the enzyme DNA ligase joins them together to form recombinant plasmids

Question 15

What is the process of introducing the vector into a host bacterial cell for replication (transformation) for ‘In Vivo’ cloning

Answer 15

• Once the required DNA has been incorporated into at least some of the plasmids, they must be reintroduced into the bacterial host
• The bacterial cells and plasmids are incubated together in a medium containing calcium ions
• The Ca2+ (and temperature changes) cause the bacterial membrane to become more permeable, allowing the plasmid to enter
• After this, not all bacterial cells will contain the recombinant DNA, so marker genes are required to identify the required plasmids

Question 16

Why don’t all bacterial cells contain the required DNA fragments after Transformation during ‘In Vivo’ cloning

Answer 16

• Only a few cells will take up a plasmid when mixed together
• Some plasmids will have closed up before incorporating the DNA fragment
• DNA fragments may have joined together to form their own plasmids

Question 17

How are marker genes used to identify the desired plasmids after ‘In Vivo’ cloning has taken place

Answer 17

A second, separate gene within the recombinant plasmid can be used to allow the required cells to be identified for cloning
- A plasmid with an identifiable characteristic is chosen, and the restriction endonuclease cuts the gene responsible for this trait when incorporating the DNA fragment
- ∴ The gene responsible for the recognisable trait will no longer work if the plasmid contains the required gene (has been cut)
- ∴ Required plasmids (bacterial cells) are those that no longer show this marker trait

Question 18

What are the main types of marker genes used in ‘In Vivo’ cloning

Answer 18

• Antibiotic resistant markers
• Fluorescent markers
• Enzyme markers

Question 19

How are Antibiotic-resistant Marker genes used to identify the desired plasmids as part of ‘In Vivo’ cloning

Answer 19

• Genes within the chosen plasmid produce enzymes that break-down antibiotics
  ∴ Bacteria with this gene will survive in the presence of the antibiotics
• eg. R-plasmid: Carries genes for resistance to two antibiotics (Ampicillin and Tetracycline)
• The gene required to form the recombinant DNA is inserted into the plasmid, with the restriction endonuclease cutting through the Tetracycline resistant gene
• ∴ The recombinant DNA will no longer be resistant to Tetracycline, but will still be resistant to Ampicillin
• This means that after incubation with the plasmids, the bacteria can be grown in medium with Ampicillin, and only those with a plasmid present will survive
• After this, the remaining bacteria can be grown in a medium with Tetracycline and a technique called ‘Replica plating’ can be used to determine which bacteria contain the recombinant plasmid, as these are the ones that don’t survive
  (replica colonies grown for cloning)

Question 20

What is the process of ‘Replica plating’ used to determine which bacteria contain the recombinant DNA through the use of antibiotic-resistant markers during ‘In Vivo’ cloning

Answer 20

• Bacteria that survived in Ampicillin (contains plasmid) is grown in Tetracycline medium
• The bacteria that die must have contained the recombinant DNA, as they are no longer resistant (Tetracycline resistant gene was cut to insert the required fragment)
• On a ‘replica plate’, only the colonies that died on the Tetracycline plate are grown
• This culture will now only contain the recombinant plasmids for use in DNA technology

Question 21

How are Fluorescent Marker genes used to identify the desired plasmids as part of ‘In Vivo’ cloning

Answer 21

• Gene from Jellyfish is transplanted into the plasmid
• Gene produced ‘green fluorescent protein’ (GFP)
  ∴ Bacteria with this gene will fluoresce
• Gene to be cloned for the recombinant DNA is inserted into the centre of the GFP gene (GFP gene cut with restriction endonucleases)
  ∴ Recombinant plasmids will no longer fluoresce
• ∴ After incubation, bacterial hosts with the recombinant DNA will no longer fluoresce, so these can be retained for cloning

Question 22

How are Enzyme Marker genes used to identify the desired plasmids as part of ‘In Vivo’ cloning

Answer 22

• Gene that produces the enzyme ‘lactase’ is transplanted into the plasmid
• Lactase will cause a colourless substrate to turn blue
• Gene to be cloned for the recombinant DNA is inserted into the centre of the lactase gene (lactase gene cut with restriction endonucleases)
  ∴ Recombinant plasmids will no longer be able to produce lactase
• ∴ After incubation, bacterial hosts with the recombinant DNA will no longer cause the colourless substrate to turn blue, so the colourless cells can be retained for cloning

Question 23

What are the advantages of ‘In Vivo’ cloning

Answer 23

• Useful if you need to introduce the gene into another organism
  
  • Once in the gene is in the plasmid, this vector can be used to transport it to another organism
  • eg. Humans, through a process called ‘gene therapy’
• Involves no risk of contamination
  
  • The gene and plasmid are cut by the same restriction endonuclease, so have complementary sticky ends
  • Any contaminant DNA won’t have complementary sticky ends, so won’t be taken up by the plasmid (won’t be cloned)
• Very accurate
  
  • Mutations are rare
  • Few errors in cloning process (much less then PCR)
• Cuts out specific genes
  
  • ∴ Very precise procedure, as the culturing of transformed bacteria produces many copies of a specific gene
• Can produce large quantities of required gene
  
  • Proteins can be produced for commercial or medical use (eg. Insulin)

Question 24

What is ‘In Vitro’ cloning

Answer 24

The process by which required DNA fragments are cloned through the ‘Polymerase chain reaction’ (PCR)

Question 25

What is required for ‘In Vitro’ cloning to take place

Answer 25

• DNA fragment (to be copied)
• DNA polymerase enzyme to join together nucleotides
  eg. ‘Taq polymerase’
• Primers
  
  • Short sequence of nucleotides
  • Complementary to the bases at one end of each of the two complementary DNA fragments
• Nucleotides
  
  • To join together and form copies of the fragment
• Thermocycler
  
  • Computer controlled machine that varies temperature precisely over a period of time, allowing the PCR to occur as an automated process

Question 26

What is ‘Taq polymerase’ and why is it used in the Polymerase Chain Reaction

Answer 26

Type of DNA polymerase enzyme (joins nucleotides together) from a Bacteria that lives in hot springs (Thermus aquaticus)
∴ Tolerant to heat (thermostable), so doesn’t denature during PCR

Question 27

What is the Process of the ‘Polymerase chain reaction’ (PCR)

Answer 27

1) Separation of the DNA strands
- DNA fragments, Primers and Taq polymerase are placed in a vessel in the Thermocycler
- Temp increased to 95°C
- ∴ 2 strands of DNA separate as hydrogen bonds break

2) Annealing (addition of primers)
- Mixture cooled to 55°C
- ∴ Primers join (anneal) to their complementary bases at the end of the DNA fragments
- This provides the starting sequence for the DNA polymerase, as it can only attach nucleotides to an existing chain
- Primers also prevent the 2 separated strands from re-joining

3) Synthesis of DNA
- Temp increased to 72°C
(Optimum temperature for the DNA polymerase to add complementary nucleotides along each strand)
- Beginning at the primers, the complementary strands are synthesised to the end of the chain

Process results in two identical copies of the original DNA, and the cycle then repeats, doubling the total number each time
(each cycle takes ~2 mins)

Question 28

What are the advantages of ‘In Vitro’ cloning

Answer 28

• Extremely rapid
  
  • Quickly produces a large number of copies from a small sample
  • Valuable in Forensics (eg. small blood sample)
• Doesn’t require living cells
  
  • Only requires the DNA base sequence to be copied
  • No complex culturing technique that take time and effort

Question 29

What is the main risk associated with the ‘Polymerase chain reaction’ (PCR)

Answer 29

Contamination
- The process of the PCR will copy and amplify all DNA that is present
- This may lead to the cloning of a potentially harmful gene, giving the process some associated risks

Question 30

What are the benefits/uses of recombinant DNA technology

Answer 30

• GM organisms (plants, animals, microorganisms) can be modified to produce useful substances
  
  • eg. Antibiotics, hormones, enzymes, etc.
  • Production is cheaper
• GM microorganisms can be used to control pollution
  
  • eg. Modified to break down oil and harmful gases
• GM crops can be engineered for financial and environmental advantages
• GM crops can help prevent disease (eg. Golden rice with added vitamin A)
• DNA technology can be used to replace defective genes (gene therapy), curing genetic disorders
• Genetic fingerprinting can be used in Forensics

Question 31

What are the risks of recombinant DNA technology

Answer 31

• It is impossible to predict the ecological consequences of GM organisms of the environment
  (‘Suicide genes’ can be added to limit the impact)
• Recombinant DNA may be passed to other organisms (eg. Viruses)
• Manipulating DNA may impact metabolic pathways
• May lead to antibiotic resistance
• Mutations may alter the engineered gene
• May impact Natural Selection (Evolution)
• May have economic consequences (eg. GM bananas that can grow in UK will impact Caribbean economies)
• May lead to genetic modification being used for political/personal advantages (eg. Designer babies, Eugenics, etc.)
• Expense may make the technology only accessible to the wealthy (eg. disease treatment)
• May be considered immoral to ‘tamper with nature’

Question 32

How are specific genes located within DNA

Answer 32

A labelled DNA probe attaches to the specific gene, through the process of ‘DNA Hybridisation’, highlighting it’s location

Question 33

What are ‘DNA probes’

Answer 33

Short, single-stranded length of DNA with an attached label to make it easily identifiable
- Strand is complementary to a specific base sequence on the DNA, so the probe can attach to and help located this gene

Question 34

What are the two common types of DNA probes used to locate a gene

Answer 34

• Radioactively labelled probes
  
  • Made up of nucleotides with the isotope P-32 attached
  • Identifiable using x-ray film
• Fluorescently labelled probes
  
  • Fluoresce under certain conditions
  • eg. when bound to target DNA sequence

Question 35

What is the process of using DNA probes to locate a specific gene or allele

Answer 35

• The base sequence of the required gene/allele is determined
  
  • Can be achieved by genetic sequencing
  • Genetic libraries contain the base sequences of most genetic diseases
• Complementary DNA fragment is produced (copied with PCR) and marker attached to form DNA probe
• The DNA sample in which the gene is being located, is heated to separate the two strands
• The DNA is then mixed with the probes, and cooled
• If the DNA contains the specific gene/allele, the complementary probe will attach (DNA hybridisation)
• The DNA is then ‘washed’ clean of any unattached probes
• The remaining hybridised DNA will be marked by the probe label, so can be identified

Question 36

What is ‘DNA Hybridisation’ and how is it carried out

Answer 36

Process by which a section of DNA/RNA is combined with a single stranded section of DNA made up of complementary bases
- First, the 2 strands of the double stranded DNA must be separated by heating (denaturation)
- When cooled, the original 2 strands will re-join
- However, if complementary DNA probes are present (small, single stranded), these are just as likely to anneal with one of the strands (DNA Hybridisation)

Question 37

What is ‘Genetic screening’

Answer 37

The process by which an Individual’s DNA is ‘checked’ to see if it contains a mutant allele that could lead to a genetic disorder
- Used to check individuals with a family history of disease, so see if they are likely to develop symptoms
- Can determine the probability of a couple having a child that has a genetic disorder
- Can detect the presence of Oncogenes, and mutated tumour suppressor genes
- Information about an individuals likelihood of developing a genetic disease can then allow informed decisions to be made about lifestyle, future treatment, etc.

Question 38

What is the process of genetic screening

Answer 38

• Hundreds of DNA probes are fixed onto a glass slide
  
  • Probes are complementary to known alleles that cause genetic disorders
• A sample of the individuals DNA is added, so that any alleles complementary to the probes will attach
• ∴ Disease causing alleles are highlighted by the probe marker
• This process allows many genetic diseases to be screened simultaneously

Question 39

How does genetic screening allow for the development of personalised medicines

Answer 39

Genetic screening allows doctors to provide advice and healthcare based on an individual’s genotype
- Depending on their gene, certain drugs may be more/less effective for each patient
- ∴ Correct drug, of correct dose, can be given to each patient
- eg. Prescribing painkiller:
- Many painkillers require an enzyme to activate them
- Genetic screening will determine if a person has the gene to produce the enzyme
- ∴ Dose can be adjusted accordingly
- eg. Vitamin E
- For people with diabetes, different genotypes mean that vitamin E either increases or decreases the risk of cardiovascular disease
- ∴ If the genotype of an individual is determined through genetic screening, the effect of vitamin E can be known

Question 40

What is ‘Genetic counselling’

Answer 40

Specific form of social work where advice and information is given to people based on family history, results of genetic screening, etc.
- Allows individuals to make decisions about themselves and their offspring
- Councillor can inform a couple of the chance of their children inheriting a certain genetic disorder, as well as the consequences of this (emotional, psychological, medical, social, economic, etc.)
- Information and the resulting course of action can help increase the chances of survival if genetic disease is detected

Question 41

What is ‘Genetic fingerprinting’

Answer 41

Diagnostic tool, used to give a specific pattern for each individual based on their unique DNA sequence
- Relies on the fact that most DNA in Eukaryotes is non-coding
- These non-coding sections are called ‘variable number tandem repeats’ (VNTR)
- The number and length of the VNTRs of an individual will produce a unique pattern through the process of Gel Electrophoresis
- More closely related individuals will have more similar VNTRs (∴ more similar genetic fingerprints)

Question 42

What is ‘Gel Electrophoresis’ and how is it carried out

Answer 42

The process used to separate DNA fragments according to their size
- DNA fragments are places on agar gel, with high voltage supplied across it
- The resistance of the gel means that larger fragments move more slowly, as the negative phosphate groups are attracted to the anode
- ∴ DNA fragments of different sizes spread out
- If fragments are labeld with radioactive probes, X-ray film can be used to locate them
- This process only works for fragments up to 500 bases long (longer must first be cut with restriction endonuclease)

Question 43

What are the 5 main stages of ‘Genetic fingerprinting’

Answer 43

1) Extraction
- DNA is separated from the cell
- Sample is usually small, so DNA quantity is increased by PCR
2) Digestion
- DNA is cut into fragments using restriction endonucleases
3) Separation
- Gel Electrophoresis is used to separate fragments of DNA according to their size
- Gel is then immersed in alkali to separate the double strands
4) Hybridisation
- DNA probes with radioactive labels then bind to specific VNTRs (targets sequences)
5) Development
- X-ray film is placed over the nylon membrane
- Radioactive probes cause a series of bars to appear at the location of the target sequence
- The pattern of these bands in unique to every individual (except identical twins)

Question 44

What are some potential uses for genetic fingerprinting

Answer 44

• Paternity tests
  
  • Half of a person’s genetic material is inherited from each parent
  • There will be corresponding bands in each of the parent’s DNA fingerprints
• Investigating genetic variability within a population
  
  • A population with more similar DNA fingerprints has less genetic diversity (Vice versa…)
• Forensic science
  
  • DNA fingerprint of suspect can be compared to that of DNA left at the crime scene
  • However, close match may not mean guilt (close relative, contamination, witness to the crime, etc)
• Medical diagnosis
  
  • DNA fingerprint of patient can be compared to that of various genetic disorders, to determine the prob of developing symptoms
  • Can also be used to determine the nature of microbial infections
• Plant and animal breeding
  
  • Can prevent inbreeding during breeding programmes
  • Can aid selective breeding by determining if an individual has a desirable allele
  • Can establish the family tree of an individual