Chapter 21 - Recombinant DNA Technology Flashcards
What are the 3 methods of isolating target genes
- Restriction enzymes
- Reverse transcriptase
- Artificially synthesising a gene
Explain how restriction enzymes can be used to isolate target genes
- DNA contains palindromic sites (eg RACECAR)
- Restriction enzymes cut DNA at specific palindromic sites called restriction sites
- If there’s a restriction site either side of a target gene restriction enzymes can be used to cut it out
- Using a restriction enzyme will leave sticky ends (unpaired bases)
Explain how reverse transcriptase can be used for isolating target genes
- Reverse transcriptase is an enzyme that conducts transcription backwards
- RNA polymerase usually used to get mRNA from DNA
- Instead reverse transcriptase is used to get cDNA (complementary DNA) for mRNA
- cDNA has no nucleus
Explain how a gene can be artificially synthesised
- A gene machine is used to make DNA from scratch
- 25 nucleotides are joined together at once
- This forms an oligonucleotide
- These can be joined to form a synthetic gene
What is meant by a vector
Something used to move DNA from one place to another
Eg plasmids
What is meant by recombinant DNA
DNA from more than one source/organism
eg plasmid and target gene
What is meant by a transgenic organism
An organism that contains recombinant DNA
Why is ice-cold calcium chloride and heat shock used
This increases the permeability of bacterial cell wall
What is a marker gene
A gene paired with a target gene that can be identified to check if the vector has been infected properly
Why do marker genes need to be used
- Vectors are often not taken up by bacteria
- Marker genes need to be used to tel which bacteria have become transgenic you need marker genes
- Transgenic bacteria contain the recombinant DNA
- These can be selected and cultures in transgenic bacteria
What marker genes are often used
UV fluorescence or antibiotic resistance
How can UV and antibiotic resistance be identified
- Flourescent under UV light
- Able to survive in a culture with the antibiotic
What is the purpose of the polymerase chain reaction (PCR)
To amplify DNA (make millions of copies)
What is PCR sometimes called
in vitro DNA amplification
What is needed for PCR
- DNA sample
- Free DNA nucleotides
- Primers are used to select which part of the DNA is copied
- DNA polymerase
What are primers
Short sequences of DNA that are complementary to the start of the DNA sample
Describe the method of PCR and explain each stage
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Summarise gene technology
- Isolate target gene
- Insert gene into vector
- Insert vector into bacteria
- Identify transgenic organisms
- Culture transgenic bacteria
- Extract and purify protein
Infolder
What is gene therapy
Changing faulty alleles that cause genetic disease
Explain an example of gene therapy for dominant alleles
Huntington’s
- Sufferer will be heterozygous
- They already have the functional allele
- Have to silence dominant allele
- Use a vector to add a DNA fragment into the dominant allele
- Dominant allele won’t be transcribed
- Recessive allele expressed
Explain an example of gene therapy for recessive alleles
Cystic fibrosis
- Sufferer will be homozygous
- Use a vector to add the functional allele to DNA
- Dominant allele will be expressed
What are two types of gene therapy
Germ line gene therapy
Somatic gene therapy
What is germ line gene therapy
Changing the alleles of gametes
Future offspring inherit changes
What is somatic gene therapy
Changing the alleles of body cells
Offspring don’t inherit changes
What are some possible problems with gene therapy
- Alleles could be inserted into the wrong locus
- Could silence wrong gene by mistake (eg tumour suppressor gene causing cancer)
- Gene could be overexpressed
- Use of gene therapy could be used for non-medical purposes eg cosmetic
What are some examples of genetically modified organisms in agriculture
Golden rice
soya beans
What are the uses of golden rice
- Gene from corn
- Put in rice
- Expresses vitamin A to reduce vitamin A deficiency which causes blindness
What are the uses of GM soya beans
- Express protein from bacteria
- Protein is toxic to insects
- Fewer insects eat soya plants
- Less need for pesticides
- More efficient food chain so less energy is wasted
What are some disadvantages of GM crops
- Create a monoculture, less diversity so susceptible to disease
- Have to buy seeds every year as second generation are infertile
- Decreases biodiversity
What is an example of how GM organisms in industry and research
- Making enzymes eg rennin
- Transformed pathogens to treat disease, they attack other pathogens but don’t infect humans
What is a disadvantage of making enzymes
Reduces energy usage and cost
What are advantages of using transformed pathogens to treat disease
- Pathogens won’t develop resistance
- Reduces suffering from disease
What are disadvantages of using transformed pathogens to treat disease
- Could mutate and infect humans
- Could be used as a bio weapon
What is an example of how GM organisms can be used in medicine
Insulin
What are some uses of genetically modified organisms for medicine
- Can transform bacteria to express proteins so make human proteins, which is cheaper and easier than synthetically making proteins
- Mammals can be genetically modified to produce useful products in their milk
What are some disadvantages of using GM organisms for medicine
- Possible unexpected problems eg cancer
- Ethical issues such as using animals as commodities
What is a DNA probe
A short sequence of DNA (with a label attached) that is complementary to a specific allele/mutation/gene
Explain how DNA probes are used
- Used to test if a sample of DNA contains a specific sequence
- Attach a label to DNA probe
- If sequence is present DNA will hybridise (form hydrogen bonds) and stick to DNA sample with a complementary sequence
- Rinse to remove unhybridised DNA probes
- View labels eg UV
- DNA must be single stranded for this to work
Describe DNA microarray
- Label attached to human DNA
- Test for multiple alleles/mutations at once
- Many DNA probes attached to a tile in a grid
- Add patients DNA with label attached
- DNA will hybridise to complementary DNA
- Rinse
- View labels
Explain how target genes can be inserted
- Isolate target gene (restriction enzymes, reverse transcriptase or gene machine)
- Add promotor region, terminator region, sticky ends and marker gene
- Insert gene into a vector by using restriction enzymes to cut plasmid
- Sticky ends are complementary
- DNA ligament enzymes reform the phosphodiester bonds
- Forming recombinant DNA
- Insert vector into bacteria
- Bacteria becomes a transgenic organism
- Use ice-cold calcium chloride and heat shock
What are some uses of DNA probes
Genetic counselling
Genetic screening
Personalised medicine
How can DNA probes be used for genetic counselling
- Identify carriers of genetic disease, specific allele and most effective treatment
- Healthcare professionals can advise people about the risks eg passing on heritable diseases, developing disease later in life and making decisions of treatments and preventions
How can DNA probes be used for genetic screening
- Parents can see if they are carriers of recessive alleles (IVF)
- Can diagnose and treat before symptoms show eg babies are screened for cystic fibrosis
How can DNA probes be used for personalised medicine
- If a doctor knows a patients genotypes, they can give them the best drugs for them
- If you know a patient has an allele that causes a side effect with the normal drug they can be given another
What is the purpose of genetic fingerprinting
Identifying individuals by comparing the differences in their variable number tandem repeats
Explain the process of how genetic fingerprinting allow you to identify individuals
- Non-coding DNA contains lots of variable number tandem repeats
- Don’t code for proteins
- Don’t effect phenotype
- Vary more than coding DNA
- The number of repeats of each VNTR varies
How can genetic fingerprinting be used for forensic use
- Samples of DNA from crime scene and suspects are needed
- Amplify DNA using PCR
- Run gel electrophoresis
- The chance of 2 individuals having the same number of VNTR’s is very low
How can genetic fingerprinting be used for relatedness
- The more closely related 2 individuals are, the higher the percentage match of VNTR’s eg paternity test =50%
- Conservation of endangered species
- Avoid inbreeding I’m small population by mating least related individuals to maintain genetic diversity
How can genetic fingerprinting by used for medical diagnosis
- Genetic fingerprints can be used to test for specific combinations of alleles
- So can be used to diagnose genetic disorders
