Gene technologies (21) Flashcards
What is recombinant DNA technology?
the transfer of DNA fragments from one organism to another
What are 2 ways we can amplify DNA fragments?
- polymerase chain reaction (in vitro)
- using host cells (in vivo)
What 4 things does the reaction mixture in the PCR contain?
- 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
What are the 3 steps of inserting a DNA fragment into a vector?
(insertion)
1) a plasmid is used as a vector
2) plasmid is cut using the same restriction enzymes as the DNA so that the ends are complementary
3) DNA ligase joins the fragment and plasmid together
What are the 5 stages of making a protein using DNA technology of a gene transfer and cloning?
1) isolation
2) insertion
3) transformation
4) identification
5) growth/cloning
Why does recombinant DNA technology work?
genetic code is universal, therefore transcription and translation occur by the same mechanism and result in the same amino acid sequence across organisms
What are the 3 steps in using reverse transcriptase to produce DNA fragments?
(isolation)
1) mRNA complementary to the target gene is used as a template
2) it’s mixed with free nucleotides, which match up to their base pairs, and reverse transcriptase
3) sugar-phosphate backbone forms and complementary DNA is created
What 2 things can you use to isolate DNA fragments?
- reverse transcriptase
- restriction endonucleases
What are the 3 steps in using enzymes to produce DNA fragments?
(isolation)
1) restriction endonucleases cut DNA at specific base sequences
2) different restriction endonucleases cut at different points, but one will always cut at the same sequence
3) therefore, using different restriction endonucleases allows you to cut out a certain gene of interest
What are the 4 steps of how the polymerase chain reaction amplifies DNA fragments?
(insertion)
1) requires DNA polymerase, nucleotides and primers
2) DNA fragments heated to 95°C to break hydrogen bonds
3) reduce temperature to allow primers to bind to strands
4) increased temperature again to activate DNA polymerase so free nucleotides can join
What are the 3 steps of inserting the vector into a host cell?
(transformation)
1) host cells (bacteria) are mixed with vectors in an ice-cold solution
2) then it’s heat shocked to encourage the cells to take up the vectors
3) cells are then grown and the DNA fragment will be cloned
What are the 3 uses of DNA probes?
- to screen someone’s DNA for a particular heritable health condition
- to identify gene for use in genetic engineering
- to predict how someone will respond to a drug
What are 2 benefits of genetic profiling?
- can identify heritable diseases very early so they can be treated before symptoms develop, reducing impact on individual
- can allow for personalisation of treatment to make it more effective
What are the 2 steps in identifying transformed cells?
(identification)
1) marker genes (e.g. that code for fluorescence) can also be inserted into vectors along with the DNA
2) as cells begin to grow, UV light can be used to identify which cells have taken up the vector and which haven’t
What are the 4 steps on how DNA probes can be used to locate specific alleles?
1) probe is designed so that its sequence is complementary to the allele you want to find
2) they’re labelled and amplified using PCR
3) then they’re added to a sample of single stranded DNA
4) the probe will bind if the allele is present
What are the 2 purposes of DNA hybridisation?
- to measure degree of difference between 2 strands of DNA
- can be used to compare someone’s DNA to a certain gene to see if they have it
What are the 2 steps of DNA hybridisation?
1) 1 DNA strand is labelled and mixed with an unlabelled comparison strand
2) the more similar the strands, the stronger they will bind so more energy that will be required to break the strands apart
What is genetic fingerprinting?
technique used to compare 2 DNA samples and determine whether they came from the same individual
What are the 2 steps on how genetic fingerprinting is able to work?
1) every organisms genome contains non-coding regions called variable number tandem repeats (VNTRs)
2) probability of 2 individuals having the same VNTRs is very low, so we can compare these areas to see if the 2 DNA samples are from the same person
What are the 4 steps of genetic fingerprinting?
1) DNA sample obtained
2) VNTRs cut out using restriction enzymes, labelled and cloned using PCR
3) fragments separated using gel electrophoresis
4) banding patterns of each sample can then be compared
What are the 3 steps on how gel electrophoresis work?
1) DNA fragments are placed at one end of a slab of gel
2) electric current is applied, causing the DNA fragment to move towards other end of the gel (shorter fragments travel further)
3) pattern of bands created is unique to ever individual
What are 3 uses of genetic fingerprinting?
- forensics e.g. to identify victims of suspects
- medical diagnosis e.g. to identify type of haemoglobin produced by an individual to diagnose sickle cell anaemia
- animal + plant breeding e.g. breed out harmful alleles and ensure pedigree
What is a DNA probe?
single strand of DNA with complementary bases
