3.8.2 Recombinant DNA Technology Flashcards
What are the stages of in vitro gene cloning
Isolation, Insertion, Transformation, Identification, Growth
How is cDNA produced
Reverse transcriptase is used to covert mRNA into double stranded cDNA.
How is cDNA different to the original nuclear gene and why might this be a good thing
cDNA would not contain any introns. THis is necessary if the gene is to be inserted into a prokaryote which does not have the ability to splice out introns
State three methods that could be used to identify recombinant bacteria from non-recombinant
Antibiotic resistance markers, fluorescent markers, enzyme markers
What is a restriction enzyme?
Enzymesused to cut double stranded DNA at a specific recognition site
How are restriction enzymes useful in producing recombinant plasmids
If the same restriction enzyme is used to cut the plamid and target gene then 2 pieces of DNA with complementary sticky ends will be produced. The overhanging or exposed bases can join up via complementary base pairing
How is DNA ligase used in DNA technology
used to join the sugar phosphate backbone after target gene and plamid anneal together
Explain how 2 antibiotic resistance genes on one plamid can be used to identify recombinant bacteria
If two resistance genes are used the target gene is inserted into one of these genes. All bacteria that have taken up a plasmid will be resistant to one antibiotic (ampicillin usually) but bacteria that DONT have resistance to the second antibiotic (tetracycline usually) have taken up the recombinant plasmid. They can be identified using replica plating
What is the function of PCR?
Used to make copies of DNA fragments, increasing the size of the sample (amplify the DNA)
What is in the reaction mixture for PCR?
DNA, buffer, primers, free nucleotides, DNA polymerase
Describe the process of PCR
1)95 deg C to separate the strands, 55 to anneal the primers, 72 for DNA polymerase to join the nucleotides
What is a DNA primer
Short, single-stranded DNA sequence complementary to the ‘end’ of a DNA fragment
Why are primers used in PCR
The DNA polymerase enzyme cannot bind to single stranded DNA but must “extend” or lengthen the
What is genetic engineering?
Genes from one organism transferred into genome of another
How are organisms genetically modified to produce drugs ?
The gene for the protein is isolated using restriction enzymes. The gene is copied using PCR. Copies are inserted into plasmids. Plasmids are transferred into microorganisms. the modified microorganisms are grown in large containers so they divide and produce lots of the useful protein, from the inserted gene. the protein is purified and used as a drug.
Give an example of a drug that is produced from genetically modifided bacteria
human insulin (type 1 diabetes) and human blood clotting factors (haemophilia)
What are the benefits of GMO?
Modify agricultural crops to give higher crops or to be more nutritious. reduces risk of famine and malnutrition. Modify crops to have pest resistance, so fewer pesticides are needed. this reduces costs and reduces any environmental probs. dont need to refrigerate vaccines produced in plant tissue. so vaccines are available to more people
What are the risks of GMO?
Transmission of genetic material between plants/ could lead to herbicide resistant weeds or crops containing drugs/Long-term impacts - unforeseen consequences/Increased chemical use in plants/Wrong to genetically modify animals purely for human benefit
State three methods that could be used to identify recombinant bacteria from non-recombinant
Antibiotic resistance markers, fluorescent markers, enzyme markers
Explain why DNA must be treated with restriction enzymes before being inserted into a plasmid.
So the DNA and plasmid have complementary sticky ends
Define ‘DNA probe’
Short, single stranded, labelled piece of DNA that is complementary to a specific sequence
what are the 2 most common types of DNA probe
Radioactive (normally using 32-P) or fluorescent
What do we mean by ‘hybridisation’?
When two strands of DNA from different sources join together (for example a DNA probe and a gene)
When and why would PCR be used in genetic fingerprinting?
To amplify the DNA before it is digested and separated
Name enzymes involved in genetic engineering and say how they are used
Restriction endonuclease - to isolate DNA, DNA ligase - inserts sections of DNA into host/plasmid, DNA polymerase - used in PCR
What happens when an electric current is passed through an electrophoresis gel?
DNA fragments are negatively charged so they move towards the positive electrode at the far end of the gel
State the first step in gel electrophoresis
DNA is placed into a well and covered in a buffer solution that conducts electricity
State 3 uses of DNA fingerprinting
Forensic science, medical diagnosis, plant and animal breeding, determining genetic variability in a population
Why do the small DNA fragments move further in gel electrophoresis?
They move faster as the current is applied
Give stages of Genetic fingerprinting
Extraction, digestion, separation, hybridisation, development
