8.4: Gene Technologies Flashcards
What is meant by recombinant DNA technology?
The transfer of DNA fragments from one organism to another
Why does recombinant DNA technology work?
Because the genetic code is universal and therefore transcription and translation occur by the same mechanism and result in the same amino acid sequence across organisms
Summarise the process of using reverse transcriptase to produce DNA fragments
mRNA complementary to the target gene is used as a template. It is mixed with free nucleotides which match up to their base pairs, and reverse transcriptase which forms the sugar-phosphate backbone to create cDNA (complementary DNA)
Summarise process of using enzymes to produce DNA fragments
Restriction endonucleases (RE) cut DNA at specific sequences. Different REs cut at different points but one RE will always cut at the same sequence. Therefore using particular REs allows you to cut out a certain gene of interest.
In which two ways can we amplify DNA fragments?
- In vitro/ polymerase chain reaction (PCR)
- In vivo/ using host cells
Describe the reaction mixture in the first stage of PCR
Contains the DNA fragments to be amplified, primers that are complementary to the start of the fragment, free nucleotides to match up to exposed bases and DNA polymerase to create the new DNA
Summarise process of amplifying DNA fragments using PCR
- Heated to break apart the DNA strands
- Cooled to allow primers to blend
- Heated again to activate DNA polymerase and allow free nucleotides to join
- New DNA acts as template for next cycle
Summarise process of inserting a DNA fragment into a vector
A plasmid (circular DNA from bacteria) is used as a vector, and is cut out using the same restriction enzymes as the DNA, so that the ends are complementary. DNA ligase joins fragment and plasmid together
Summarise process of inserting a vector into the host cell
Known as cell transformation. The host cells (bacteria) are mixed with the vectors in an ice-cold solution, then heat shocked to encourage the cells to take up the vectors. The cells can then be grown and the DNA fragments will be cloned
Summarise process of identifying transformed cells
Marker genes e.g. coding for fluorescence can also be inserted into vectors along with DNA. When cell begins to grow, UV light can be used to identify which cells have taken up the vector and which havent
How can DNA probes be used to locate specific alleles?
The probe is designed so that its sequence is complementary to the allele you want to find. They are labelled, amplified using PCR and then added to a sample of single stranded DNA. The probe will bind if the allele is present.
Give some applications of DNA probes
- To screen someone’s DNA for a particular heritable health condition
- To identify a gene for use in genetic engineering
- To predict how someone will respond to a drug
What is the purpose of DNA hybridisation?
To measure the degree of difference between two strands of DNA. Can be used to compare someone’s DNA to a certain gene to see if they have it.
Summarise process of DNA hybridisation
One DNA strand is labelled and mixed with an unlabelled comparison strand. The more similar the strands, the more strongly they will bind and more energy will be required to break the strands apart.
What are the benefits of genetic profiling?
Can identify heritable diseases very early, and therefore begin to treat them before symptoms develop, reducing impact on individual. Treatment can be personalised to make it more effective
