Gene technology Flashcards
(38 cards)
What is PCR used for?
PCR is used to amplify target DNA sequences that are present in a DNA source
Describe the process of PCR
- DNA sample to be amplified is heated to 95ºC - break the H bonds between strands
- The mixture is cooled to 40-60ºC which allows primers to anneal
- Taq polymerase and free nucleotides added, the mixture is heated to 70ºC. Taq polymerase copies each stand and the process is repeated.
What are the functions of primers
- Stop the 2 DNA strands rejoining
- Bracket the section of DNA to be copied
- DNA replication can only start with a double stranded region
What is the role of PCR
Forensics, fingerprinting
What are DNA probes used for?
Identify sections of DNA that contain a specific sequence of bases. Once the specific section is identified, restriction endonuclease enzymes cut out the required section
What is a probe?
A probe is a short single strand of DNA with a specific base sequence that will hybridise to the complementary target section if present.
Describe the process of genetic fingerprinting
- Extracted from a DNA sample often PCR used to amplify the same
- The DNA is cut into fragments of different sizes by restriction enzymes. MRSs the number of repeats differs
- Fragments separated by gel electrophoresis
- Heat treated to make it single stranded
- Transferred to a nylon membrane and radioactive/fluorescent probes added.
- The labelled DNA is added to a film and is recognisable as a genetic finger print
What is the role of genetic finger printing in society?
- Each individual has unique MRSs which means you can identify people
- Establish ancestry paths
Describe restriction endonucleases
- They occur naturally as defence bacterial enzymes that cut up foreign DNA injected by bacteriophage
- Cuts the DNA via a hydrolysis reaction at a specific section of bases called a hydrolysis site
- Can either create blunt or sticky ends
What are sticky ends?
Short sections of DNA where there is only one strand and the bases in that section are unpaired and exposed.
What is the role of reverse transcriptase?
It is used to make the desired section of DNA from the genes mRNA
How does reverse transcriptase work?
- Isolating and extracting the mRNA of the desired gene
- Reverse transcriptase can be used to make a single strand of DNA using mRNA as a template.
- The enzyme DNA polymerase is used to make double stranded DNA from cDNA
How can differences in the nucleotide sequence be identified?
Gene sequencing and genetic markers
What are two types of genetic markers?
- MRSs - Microsatellite Repeat Sequences, junk DNA has short tandem repeats, the number of repeats is unique to each individual
- SNPs - Single Nucleotide Polymorphisms, A change in one nucleotide in a section of DNA can give rise to different alleles.
Describe plasmids as vectors
The bacterial plasmid is a closed loop of DNA. If cut with the same restriction endonuclease enzyme used to cut the donor DNA complementary stick ends will be made. They will match up perfectly. DNA ligase will join the two strands together due to the formation of phospho-diester bonds. The donor DNA is spliced into the plasmid and it becomes a closed loop again. The plasmid is now a recombinant plasmid.
Describe viruses as vectors
Viruses are naturally adapted to shoot their genetic material into a host cell. A bacteriophage can have donor DNA spliced into its own which is then fired into the host cell.
How do you insert the gene into the host cell via plasmids
- Bacterial cells incubated with Calcium ions and subjected to heat shock 0ºC to 40ºC - makes bacteria more permeable
- Identify cells that have taken up the desired Gene - use of marker genes e.g. (1) antibiotic resistance, cuts one gene for antibiotic resistance using replica plating, Replica plating blotting the original plate, the pressing onto a new plate. (2) Using gene probes - identify if host DNA is recombinant
What are some genetically engineered microorganisms
- Insulin - for diabetes, bacteria. Previously had to be extracted from dead animals
- HGH, Enzymes, Adhesives, lung surfactant protein and interferon
How are virus’ used to target cancer cells?
Genetically modified so that they do not harm normal cells, provides a stronger immune response
How do viruses treat bacterial infections?
When bacteria becomes resistant to antibiotics, GM viruses can support antibiotic action by disrupting these evolving defensive strategies in antibiotic resistant bacteria
How do transgenic plants come about?
- Agrobacterium enter the plant at a point where it was damaged
- Gene guns - coat microscopic pellets with donor DNA which is then fired into the host cell. Hit or miss
What are benefits of GM crops?
- Herbicide resistance
- Pesticide resistance
- Disease resistant
- Greater ecological range - drought tolerant
- Nutritional enhancement
What are downfalls of GM crops?
- Not natural
- Creation of super weeds
- Outcompete non enhanced species
- Can cause allergies
How is donor DNA incorporated into animals?
- Use of liposomes - use of a lipid carrier allows it to cross the lipid bilayer
- Electroporation - Disrupt cell membrane with high voltage
- Viruses
- Insert host DNA directly into the fertilised eggs of an animal
