Biology 1002B Study Guide - Final Guide: Channelrhodopsin, Restriction Site, Restriction Enzyme

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19 Apr 2017
Cycle : DNA Technologies
Basic mechanism of propagation of action potentials in neurons
1. At electrical resting potential (-70 mV) due to ions
inside and outside the neuron
2. Stimulation -> ions flow in/out of the membrane to
produce an action potential (+30 mV)
Because the membrane is permeable to Na+
and K+
3. Back to rest, refractory period and original
distribution of ions is restored
All we need to do to affect nerve transmission in the brain is
affect the permeability of the membrane
How channelrhodopsin can control action potentials in neurons
o When channelrhodopsin is hit by light, it opens
If you shine a light on a neuron containing channelrhodopsin, ions are able to flow
in/out to depolarize the membrane -> we can depolarize specific neurons in the brain
Possible sources of genes for use in making transgenic organisms
1. Extract genomic DNA in Chlamy (all of its genes)
Cut the genes up in specific places with restriction enzymes
Some restriction enzymes leave sticky ends that attach to
other DNA
Produces a recombinant DNA molecule
2. Extract RNA from Chlamy and then reverse transcription
Then, allow DNA polymerase to build the double strand
Leads you with a double stranded cDNA
There are no introns or promoters/regulators from this method
3. Put sequence into a DNA printer and print the genes
Limitation: they can only print relatively short sequences
Role of restriction endonuclease enzymes in creating recombinant DNA
Restriction endonuclease enzymes: recognize restriction sites cuts the DNA at
specific locations within those sequences
o Produces restriction fragments
o There are hundreds of these enzymes with their own unique restriction site
Restriction sites: specific DNA sequences ~4-8 bp
o Most are symmetrical they read the same way in both directions
Bacteria add methyl groups to bases on their restriction sites so it knows not to
break down its own DNA when defending against viral attacks
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Basic mechanism for creation of cDNA
1. Isolate mRNA
2. Add a primer of T DNA nucleotides (dT)
Base-pairs with the poly(A) tail
3. Reverse transcriptase synthesizes a DNA copy
Produces a hybrid nucleic acid molecule (mRNA base-paired
with DNA)
Reverse transcriptase is made by retroviruses
4. RNAase enzyme degrades the mRNA
Leaves the single stranded DNA
5. DNA polymerase synthesizes the second strand
Produces the double-stranded complementary DNA (cDNA)
6. Add restriction sites to each end of the cDNA and insert into a cloning
Limitation of cDNA: it only includes copies of genes that were active
o Can also be advantageous if you want to identify the genes active in one
cell type and not another
Advantages of cDNA:
o Provides clues to the changes in gene activity responsible for cell differentiation
o Already has introns removed -> bacteria can transcribe/translate it to make eukaryotic proteins
Role of plasmid (expression) vectors in genetic engineering
Expression vector: DNA molecule with added elements necessary for gene transcription and translation
into which DNA fragment can be inserted
o Prokaryotes: promote, ’ (SD o ad ’ UTR, stat ad stop odo, tasiptio teiato
o Eukaotes: egulato seuees, poote TATA o, ’ ad ’ poladelatio sigal UTR,
start and stop codon
o Contains 2 genes useful for distinguishing between those with/without the recombinant plasmid
E.g. ampR gene encodes an enzyme that breaks down the antibiotic ampicillin
Those with the vector are ampicillin resistant
E.g. lacZ+ gene encodes for ß-galactosidase that hydrolyzes lactose
The recombinant DNA disrupts the gene -> no ß-galactosidase
ß-galactosidase turns Xgal blue (blue-white screening)
Cloning a gene:
1. Isolate genomic DNA and digest with restriction enzyme
2. Digest plasmid cloning vector with the same enzyme
3. Mix the 2 and ligate with DNA ligase
Produces a mixture of recombinant plasmids, non-recombinant plasmids (resealed
cloning vectors with no inserted DNA), and joined-together pieces of genomic DNA with
no cloning vector
4. Transform DNA into E. coli
Some will take up the plasmid, some will not
5. Selection: spread bacteria on medium with ampicillin and X-gal then incubate
Only those with the plasmid will survive (they have ampR)
6. Screening: X-gal distinguishes between recombinant and non-recombinant plasmids
Blue-white screening blue is non-recombinant, white is recombinant
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