chapter two: DNA manipulation techniques and applications Flashcards
define endonuclease
endonuclease are enzymes that cut DNA at a specific sequence known as the recognition/restriciton site
what are sticky and blunt ends and
sticky ends are short sections of single-stranded DNA that result from cutting DNA with an endonuclease.
blunt ends result from endonuclease cutting the 2 strands of DNA molecule at points DIRECTLY OPPOSITE each other to produce cut end
what are the uses of sticky ends and blunt ends?
sticky ends are useful for moving or adding DNA
blunt ends are useful when silencing genes or cutting DNA for electrophoresis
define the role of DNA ligase in genetic engineering
DNA ligase is the molecule glue that bonds the 2 fragments of DNA together
how do bacteria use CRISPR-CAS9?
Bacteria, when infected with a virus will be able to identify that they have been infected as CRISPR stores any viral information
Once identified, Cas9 will cut that section of DNA, allowing for the gene of the virus to not be expressed
what does CRISPR stand for?
Clustered regularly interspaced short palindromic repeats
what is CRISPR and Cas-9
CRISPR is the library of viral infections while cas-9 is the endonuclease that cuts DNA at a desired restriction site that is programmable
outline the process CRISPR-cas9 uses to cut specific DNA
- a bacteriophage attaches to the outside of the bacterial cell and injects its DNA into the cytoplasm
- sequences from the CRISPR clusters in the bacterial genome are transcribed into guide RNA
- gRNA forms a complex with the Cas9 enzyme
- gRNA/Cas9 complex docks with bacteriophage DNA at the PAM site
- Cas9 unzips the bacteriophage DNA. if gRNA is complementary witn the DNA, it binds with the complementary sequence on the DNA
- Cas9 acts as molecular scissors and cleaves the DNA several nucleotides from the PAM site
describe how CRISPR-Cas9 can be programmed to cut any DNA sequence
scientists can personally program the genetic sequence of bacteria DNA by collecting free-floating nucleotides and sticking them together with DNA ligase
using that generically made sequence, scientists can insert that ‘viral’ made code into CRISPR and any DNA sequences with that code, the CAS-9 will cut
outline the process of PCR (3 steps)
- denature - DNA is heated at 90°C+ for two minutes
- annealing - primers are added initiating DNA synthesis at 55°C for 2 minutes
- extension - taq polymerase starts at the primers and extend them using free nucleotides, forming 2 complete double strands at 72°C for one minute
state how electrophoresis can separate DNA based on size
DNA is loaded into a gel that acts like a sieve to allow smaller DNA fragments through more quickly than larger.
(smaller fragments move further down the gel compared to larger fragments)
how to run a gel electrophoresis
- DNA sample is combined with DNA loading dye.
- the mixture is placed in a well at one end of the agarose gel.
- the agarose gel is immersed in a buffer solution (salt solution).
- it is then exposed to an electric field with the positive(+)pole at the far end and the negative(−) pole (cathode) at the well
- smaller fragments move through the a agarose gel faster compared to larger fragments.
- these fragments appear as bands on a gel which can be interpreted in various ways. This usually needs to be observed under UV light.
what is a plasmid
circular, double stranded DNA that can reproduce independently
they have multiple recognition sites
process of inserting a gene into a plasmid (making a recombinant plasmid)
- cut plasmid DNA with endonuclease
- cut foreign DNA with the same endonuclease
- mix plasmids and foreign DNA
- add DNA ligase which joins the sticky ends together
- same recognition site allows same restriction enzyme to cut both the plasmid and the gene of interest
- produces matching sticky ends on the plasmids and the gene of interest
- plasmids will then joinwith the DNA of the gene of interest to form recombinant plasmids as they have complementary sticky ends.
- the two fragments can then be successfully joined by DNA ligase.
how can gel electrophoresis can be used to determine if a plasmid is recombinant
recombinant plasmids have more than one different fragment of DNA
compared to the original, recombinant plasmids have more fragments
will appear as more bands in electrophoresis
compare GMO and transgenic
GMO
- A genetically modifies organisms are organisms that have had their DNA directly manipulated
transgenic
- transgenic organisms are a subset of GMOs that have DNA from different species
examples of how genetic modification can increase crop productivity
- roundup ready canola
- canola plants are used to make canola oil
- roundup is a herbicide
- roundup ready canola has a bacterial gene inserted
- this allows the canola plant to make an enzyme to break down the herbicide roundup
- GM canola crops can be sprayed with the herbicide and only the weeds die, leaves the GM canola crops unaffected
examples of how genetic modification can provide resistance to pests or disease
- Bt cotton has a bacterial gene that allows it to produce chemicals that will kill insects
- the crops are not eaten by insects → resulting in more cotton production
- crops do not need to be sprayed with insecticide
compare genetic modification to other methods
- faster (traditional techniques like artificial selection took time to breed the organisms with the favourable traits)
- more precise (edit specifically the DNA for the trait desired)
