genetic 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
- DNA code is universal
- transcription and translation occur by the same mechanism and result int he same amino acid sequence across organisms
summarise the process of using reverse transcriptase to produce DNA fragments
(side note: reverse transcriptase makes dna copies from mrna)
- mRNA complementary to the target gene is used as a template
- mixed with free nucleotides which match up to their base pairs via complementary base pairing
- reverse transcriptase which forms the sugar-phosphate backbone is used to create cDNA (complementary DNA)
summarise process of using enzymes to produce DNA fragments
- restriction endonucleases cut DNA at specific sequences
- different endonucleases cut at different points but one endonuclease will always cut the same sequence
- so using particular endonucleases allows you to cut out a certain gene of interest
in which 2 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 complementary to the start of the fragment
- free nucleotides to match up to exposed bases
- DNA polymerase to create the new DNA
summarise the process of amplifying DNA fragments using PCR
- heated to break apart the DNA strands
- cooled to allow primers to bind
- heated again to activate DNA polymerase and allow free nucleotides to join
- new DNA acts as a template for 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 sticky ends are complementary
- DNA ligase joins the fragment and plasmid together
summarise the process of inserting a vector into a host cell
- cell transformation = the host cells are mixed with the vectors in an ice-cold solution
- then heat shocked to encourage cells to take up the vectors
- the cells are grown and the DNA fragment will be cloned
summarise the process of identifying transformed cells
- marker genes (eg coding for fluorescence) can be inserted into vectors along with the DNA
- when cells begin 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 allocate specific alleles?
- probe is designed so that its sequence is complementary to the target allele
- they are labelled, amplified using PCR and then added to a sample of single stranded DNA
- probe will bind if allele is present
list 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 the process of DNA hybridisation
- 1 DNA strand is labelled and mixed with an unlabelled comparison strand
- the more similar the strands, the more strongly they will bind and the 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 the impact on the individual
- treatment can be personalised to make it more effective
