gene tech

Question 1

What is recombinant DNA technology?

Answer 1

Transfer of DNA fragments from one organism or species, to another

Question 2

Explain why transferred DNA can be translated within cells of recipient
(transgenic) organisms

Answer 2

1. Genetic code is universal
2. Transcription and translation mechanisms are universal

Question 3

Describe how DNA fragments can be produced using restriction enzymes

Answer 3

1. Restriction enzymes cut DNA at specific base ‘recognition
   sequences’ either side of the desired gene
   ○ Shape of recognition site complementary to active site
2. Many cut in a staggered fashion forming ‘sticky ends’ (single stranded overhang)

Question 4

Describe how DNA fragments can be produced from mRNA

Answer 4

1. Isolate mRNA from a cell that readily synthesises the protein coded for by the desired gene
2. Mix mRNA with DNA nucleotides and reverse transcriptase → reverse transcriptase uses
   mRNA as a template to synthesise a single strand of complementary DNA (cDNA)
3. DNA polymerase can form a second strand of DNA using cDNA as a template

Question 5

Suggest two advantages of obtaining genes from mRNA rather than directly
from the DNA removed from cells

Answer 5

● Much more mRNA in cells making the protein than DNA → easily extracted
● In mRNA, introns have been removed by splicing (in eukaryotes) whereas DNA contains introns
○ So can be transcribed & translated by prokaryotes who can’t remove introns by splicing

Question 6

Explain how DNA fragments can be amplified by PCR

Answer 6

1. Mixture
   heated to 95oC
   ● This separates DNA strands
   ● Breaking hydrogen bonds between bases
2. Mixture
   cooled to 55oC
   ● This allows primers to bind to DNA fragment
   template strand
   ● By forming hydrogen bonds between
   complementary bases
3. Mixture
   heated to 72oC
   ● Nucleotides align next to complementary
   exposed bases
   ● DNA polymerase joins adjacent DNA
   nucleotides, forming phosphodiester bonds

Question 7

Explain the role of primers in PCR

Answer 7

● Primers are short, single stranded DNA fragments
● Complementary to DNA base sequence at edges of region to be copied / start of desired gene
● Allowing DNA polymerase to bind to start synthesis (can only add nucleotides onto pre-existing 3’ end)
● Two different primers (forward and reverse) are required (as base sequences at ends are different)

Question 8

Summarise the steps involved in amplifying DNA fragments in vivo

Answer 8

1. Add promoter and terminator regions to DNA fragments
2. Insert DNA fragments & marker genes into vectors (eg. plasmids) using
   restriction enzymes and ligases
3. Transform host cells (eg. bacteria) by inserting these vectors
4. Detect genetically modified (GM) / transformed cells / organisms by identifying
   those containing the marker gene (eg. that codes for a fluorescent protein)
5. Culture these transformed host cells, allowing them to divide and form clones

Question 9

Explain the role of enzymes in inserting DNA fragments into vectors

Answer 9

1. Restriction endonucleases / enzymes cut vector DNA
   ○ Same enzyme used that cut the gene out so vector DNA & fragments
   have ‘sticky ends’ that can join by complementary base pairing
2. DNA ligase joins DNA fragment to vector DNA
   ○ Forming phosphodiester bonds between adjacent nucleotides

Question 10

Describe how host cells are transformed using vectors

Answer 10

● To allow detection of genetically modified / transgenic cells / organisms
○ If marker gene codes for antibiotic resistance, cells that survive antibiotic exposure = transformed
○ If marker gene codes for fluorescent proteins, cells that fluoresce under UV light = transformed
● As not all cells / organisms will take up the vector and be transformed

Question 11

Suggest how recombinant DNA technology can be useful

Answer 11

Medicine
● GM bacteria produce human proteins (eg. insulin for type 1 diabetes) → more ethically
acceptable than using animal proteins and less likely to cause allergic reactions
● GM animals / plants produce pharmaceuticals (‘pharming’) → cheaper
● Gene therapy (see below)

Agriculture ● GM crops resistant to herbicides → only weeds killed when crop sprayed with herbicide

● GM crops resistant to insect attack → reduce use of insecticide
● GM crops with added nutritional value (eg. Golden rice has a precursor of vitamin A)
● GM animals with increased growth hormone production (eg. Salmon)
Industry ● GM bacteria produce enzymes used in industrial processes and food production

Question 12

Describe gene therapy

Answer 12

● Introduction of new DNA into cells, often containing healthy / functional alleles
● To overcome effect of faulty / non-functional alleles in people with genetic disorders eg. cystic fibrosis

Question 13

Suggest some issues associated with gene therapy

Answer 13

● Effect is short lived as modified cells (eg. T cells) have a limited lifespan → requires regular treatment
● Immune response against genetically modified cells or viruses due to recognition of antigens
● Long term effect not known - side effects eg. could cause cancer
○ DNA may be inserted into other genes, disrupting them → interfering with gene expression

Question 14

Suggest why humanitarians might support recombinant DNA technology

Answer 14

● GM crops increase yields → increased global food production → reduced risk of famine / malnutrition
● Gene therapy has potential to cure many genetic disorders
● ‘Pharming’ makes medicines available to more people as medicines cheaper

Question 15

Suggest why environmentalists and anti-globalisation activists might
oppose recombinant DNA technology

Answer 15

● Recombinant DNA may be transferred to other plants → potential herbicide resistant ‘superweeds’
● Potential effects on food webs eg. affect wild insects → reduce biodiversity
● Large biotech companies may control the technology and own patents

Question 16

What are DNA probes?

Answer 16

● Short, single stranded pieces of DNA
● With a base sequence complementary to bases on part of a target allele / region
● Usually labelled with a fluorescent or radioactive tag for identification

Question 17

Suggest why DNA probes are longer than just a few bases

Answer 17

● A sequence of a few bases would occur at many places throughout the genome
● Longer sequences are only likely to occur in target allele

Question 18

What is DNA hybridisation?

Answer 18

● Binding of a single stranded DNA probe
to a complementary single strand of DNA
● Forming hydrogen bonds / base pairs

Question 19

Explain how genetic screening can be used to locate specific alleles of genes

Answer 19

1. Extract DNA and amplify by PCR
2. Cut DNA at specific base sequences (either side of target gene) using restriction enzymes
3. Separate DNA fragments / alleles (according to length) using gel electrophoresis
4. Transfer to a nylon membrane and treat to form single strands with exposed bases
5. Add labelled DNA probes which hybridise / bind with target alleles (& wash to remove unbound probe)
6. To show bound probe, expose membrane to UV light if a fluorescently labelled probe was used
   OR use autoradiography (expose to X-ray film) if a radioactive probe was used

Question 20

What is gel electrophoresis?

Answer 20

● A method used to separate nucleic acid (DNA / RNA) fragments OR proteins
● According to length / mass (number of bases / amino acids) AND charge (DNA is negatively charged due to phosphate groups and protein charge varies based on amino acid R groups)

Question 21

Explain how gel electrophoresis can be used to separate DNA fragments

Answer 21

DNA samples loaded into wells in a porous gel and covered in buffer solution (which conducts electricity)
2. Electrical current passed through → DNA is negatively
charged so moves towards positive electrode
3. Shorter DNA fragments travel faster so travel further