Recombinant DNA 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 the process of using enzymes to produce DNA fragments
Restriction endonucleases cut DNA at specific sequences. Different RE’s 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
In vivo - using host cells
Describe the reaction mixture in the first stage of PCR
Contains the DNA fragment to be amplified, primers that are complimentary ti the start of the fragment, free nucleotides to match up to exposed bases and DNA polymerase to create the new DNA
Summarise the process of amplifying DNA fragments using PCR
- Heat to 72c to break apart the DNA strands
- Cool to 55c to allow primers to bind
- Heat to 96c to activate DNA polymerase and allow free nucleotides to join
- New DNA acts as template for the next cycle
Summarise the process of inserting a DNA fragment into a vector
A plasmid is used as the vector and is cut using the same restriction enzymes as the DNA so that the ends are complementary. DNA ligase joins the fragment and plasmid together
Summarise the process of inserting a vector into a host cell
The host cells 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 fragment will be cloned
Summarise the process of identifying transformed cells
Marker genes e.g coding for fluorescence can also be inserted into vectors along with DNA. When cells begin to grow, UV light can be used to identify which cells have or haven’t taken up the vector
How can DNA probes be used to locate specific alleles?
The probe is designed so that its sequence is complimentary to the allele you want to find. They are labelled, amplified using PCR, then added to a sample of single stranded DNA. The probe will bind if the allele is present
Give some application 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 if they have it
Summarise the 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 the individual. Treatment can also be personalised to make it more effective
