gene tech Flashcards
(21 cards)
What is recombinant DNA technology?
Transfer of DNA fragments from one organism or species, to another
Explain why transferred DNA can be translated within cells of recipient
(transgenic) organisms
- Genetic code is universal
- Transcription and translation mechanisms are universal
Describe how DNA fragments can be produced using restriction enzymes
- Restriction enzymes cut DNA at specific base ‘recognition
sequences’ either side of the desired gene
○ Shape of recognition site complementary to active site - Many cut in a staggered fashion forming ‘sticky ends’ (single stranded overhang)
Describe how DNA fragments can be produced from mRNA
- Isolate mRNA from a cell that readily synthesises the protein coded for by the desired gene
- Mix mRNA with DNA nucleotides and reverse transcriptase → reverse transcriptase uses
mRNA as a template to synthesise a single strand of complementary DNA (cDNA) - DNA polymerase can form a second strand of DNA using cDNA as a template
Suggest two advantages of obtaining genes from mRNA rather than directly
from the DNA removed from cells
● 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
Explain how DNA fragments can be amplified by PCR
- Mixture
heated to 95oC
● This separates DNA strands
● Breaking hydrogen bonds between bases - Mixture
cooled to 55oC
● This allows primers to bind to DNA fragment
template strand
● By forming hydrogen bonds between
complementary bases - Mixture
heated to 72oC
● Nucleotides align next to complementary
exposed bases
● DNA polymerase joins adjacent DNA
nucleotides, forming phosphodiester bonds
Explain the role of primers in PCR
● 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)
Summarise the steps involved in amplifying DNA fragments in vivo
- Add promoter and terminator regions to DNA fragments
- Insert DNA fragments & marker genes into vectors (eg. plasmids) using
restriction enzymes and ligases - Transform host cells (eg. bacteria) by inserting these vectors
- Detect genetically modified (GM) / transformed cells / organisms by identifying
those containing the marker gene (eg. that codes for a fluorescent protein) - Culture these transformed host cells, allowing them to divide and form clones
Explain the role of enzymes in inserting DNA fragments into vectors
- 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 - DNA ligase joins DNA fragment to vector DNA
○ Forming phosphodiester bonds between adjacent nucleotides
Describe how host cells are transformed using vectors
● 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
Suggest how recombinant DNA technology can be useful
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
Describe gene therapy
● 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
Suggest some issues associated with gene therapy
● 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
Suggest why humanitarians might support recombinant DNA technology
● 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
Suggest why environmentalists and anti-globalisation activists might
oppose recombinant DNA technology
● 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
What are DNA probes?
● 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
Suggest why DNA probes are longer than just a few bases
● A sequence of a few bases would occur at many places throughout the genome
● Longer sequences are only likely to occur in target allele
What is DNA hybridisation?
● Binding of a single stranded DNA probe
to a complementary single strand of DNA
● Forming hydrogen bonds / base pairs
Explain how genetic screening can be used to locate specific alleles of genes
- Extract DNA and amplify by PCR
- Cut DNA at specific base sequences (either side of target gene) using restriction enzymes
- Separate DNA fragments / alleles (according to length) using gel electrophoresis
- Transfer to a nylon membrane and treat to form single strands with exposed bases
- Add labelled DNA probes which hybridise / bind with target alleles (& wash to remove unbound probe)
- 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
What is gel electrophoresis?
● 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)
Explain how gel electrophoresis can be used to separate DNA fragments
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
