Biotechnology COPY Flashcards
Recombinant DNA Technology
Recombinant DNA technology involves the joining of different kinds of DNA
Depends on restriction enzymes and plasmids
Recombinant DNA Technology 2
involves joining DNA from different sources, it is the combining in recombinant that is helpful in remembering it, so it may have DNA from a human and DNA from Ecoli so from two different sources*
the technology depends a lot on restriction enzymes for cutting DNA and plasmids as a way to move the new DNA into a new cell*
- you are putting dna together from difference sources and you are doing this by enzymes*
Restriction enzymes
Restriction enzymes are from bacteria, very specific molecular cutting tools usually cut at a palindromic site*
Normally digest bacteriophage DNA as a defense mechanism
Each restriction enzyme is an endonuclease that cuts DNA at a specific sequence
Restriction site is usually palindromic (e.g., 5’-GAATTC-3’)
Restriction digest leaves “sticky” ends that pair with complementary ends
With ligase, can paste together two pieces of DNA cut with same restriction enzyme
When you cut with restriction enzymes makes a jaggidy cut on green DNA, the grey dna source maybe cut out of another human genome, and that grey dna represents a gene for insulin or whatever* secret here is that the green dna or grey dna is cut with the same restriction enzymes they will have complimentary ssticky ends, they have potential then to hydrogen bond and stick together in teh way that is being shown with grey stuck in green areas** piee at bottom with some green and some grey is a piece of recombinant DNA***
Enzymes naturally occuring in bacteria, defend bacteria aganist viruses because they chop up dna and we are more interested in them for a tool for biotech** they are used for precision scissors** imagine green dna is part of a plasmid, great for storing nucelic acid***
palindrome strand
same left to right as right to left
“Madam I am adam”
only see palindrome if consider two strands, so left to right on oen strand and right to left on teh other strand in that same region* on MCAT when they giveyou DNA sequences and they ask you to identify a restriction site, they are always asking you to find a palindrome, nt every restriction site in life is palindromic but on MCAT taht is what they are intersted in*
Restriction enzymes 2
- cuts at a specific site on both strands for ex btw A and G
- get sticky ends, sticky ends because can hydrogen bond to complimentary letters, idea is if take same restriction enzymes and cut some other piece of DNA with it, you woud get a fragment that has complimentary sticky ends, to draw what that may look like you may have a piece that can fit in there
- long overhang pieces are the same so can fit together like a puzzle piece; the purple piece is a common example if imagine green is DNA in an ecoli. bacterium and hte purple is the human gene for insulin, so for instance you can take an ecoli bacterium cut it with a restriction enzymes and then you can take the human genome, cut it with the same restriction enzyme and cut it with a chunk that includes the gene for insulin and then you just happen to have these cut sites around it, and if that is the cse* then this combined piece; represents recombinant dna human gene for insulin is inserted into bacterial dna* why you want to do this is you have bacterial cells that will produce insulin human protein and this is actually how most insulin as people with diabetes use is produced, they are factories that have tanks of recombinant bacteria and the bacteria has been modified in exactly this way so that each cell contains a human gene for insulin so those cells that is what hte factory is doing, the bacterial cells are pumping out insulin all day long and that insulin is gathered up and purified and packaged for people with diabetes*
- the idea is that if they are cut with molecular scissors all cut with same restriction enzymes so have ends complimentary to eachother and have ends that will fit together*
- so cut with restriction enzymes, so complimentary sequences can find eachother and then last thing you need is ligase to seal the fragments together so idea is that if you want to put a new piece of DNA into a bacterial cell, the easiest way to do this is to cut (if keep going with insulin ex) insert into bacterial plasmid*** so bacterial cells have plasmid which is another loop of dna in this case will be a piece of recombinant DNA and has its main chromosome
- recombinant plasmid containing oru gene of interest, in this case the gene of insulin, once above cutting is done and recombinant plasmid exists it can be put into bacterial cell!
Gene cloning 1
top part of diagram makign recombinant plasmid
bottom part is how it is put into a cell
having a bacteria cell that has recombinant plasmid can be used to make a protein, would go great in a factory for humn insulin, if you had a bunch of these all day long they would pump out insulin; so now when you have this bacterial cell drawn here, part of its DNA besides its main chromosome of bacterium it also has this other loop of DNA inside of hte bacterial cell, the bacterial cell will express those genes also** so recombinant plasmid containing the gene for insulin we can say the cell will express this dna and make insulin* let bacteria divide and everything and replicates and then you have thousands of cells all with thsi recominant plasmid, the plasma that has the gene for human insulin. You can use the plasmid as a way for storing that extra dna for studying
so you can use this to produce insulin like an assembly line, you can also use plasmids to store DNA** so storing DNA is kind of tricky it is not that easy to work with DNA but you can put it if you have a fragment you want to study later or a lot of fragments you want to study can use fragmetns as shelving unit, put this method of fragments in plasmid and comeback to them later* Can get them to pump out insulin and sell it to ppl who has diabetes, how insulin is made for ppl with diabetes factories that consist of big vats containing cells made exactly this way, plasmid is mostly bacterial dna, but gene for human insulin is in there so let bacteria divide everything gets copied and then you get hte cells to express the gene* so the cells, these bacteria cells start acting as the assembly line and they make all this insulin you can harvest and cell. Cells code for protein so make protein and can use it
recombinant dna= dna mistched and matched fron human and bacteria, to get it from human have to use a restriction enzyme to cut it out* if cut the human dna and hte bacteria dna with the same restriction enzymes have jaggidy sticky ends that will be complimentary to eachother* so that is the key whtever the two things are that are together cut them out fo their locations usign the same restriction enzymes
so when we store a lot of DNA in plasmids, it is called a DNA library*
Genomic library
Genomic library = segments of initial genome carried by plasmids in cloned cells
Can contain all DNA from original genome
the difference btw this and other library is has ALL THE genes vs mrna obviously not all genes?
like genomic library of chimp genome, or human genome can compare sepcies and their dna by comparing everything in the genomic libraries, becuase itsi basically like everything in the genome of the organism. ABOUT STORING DNA*
“carried by plasmids in cloned cells”= when you put a gene into a plasmid, cut plasmid with restriction enzyme cut gene with restriction enzyme and allow them to stick together/put together*
now this whole thing is called recombinant plasmid, recominant word just suggests there is dna there from two original soruces adn in this case the plasmid DNA and green dna* from human cell, now put recominanbt plasmid into a cell, now refered to as a recombinant bacteiral cell saying it is carrying a plasmid, or it contains a palsmid and as the cell divides and makes copies you get copies of the plasmid –> from library point of view this is a way of storing DNA, plasma are storing units, can popul a piece of dna in it to store it and then can study it so can use to study* the gene of interest* so that is one optionto make protein from gene of interst, if gene for insulin flwing path to the right what happens is these cells are used to produce insulin on a mass scale, so they are stimulated to do transcription/translatopon of that gene and that results in a lot of insulin being pumped out
Complementary (cDNA) library
complimentary DNA is made from mRNA*** using reverse transcriptase, cDNA by definition was dna made using reverse transcriptase from mRNA**
Complementary (cDNA) library made from mRNA (using reverse transcriptase); what we are saying now is that our starting material was mRNA in a cell and gathered up at a particular moment in time the RNA was collected and then reversed transcribed to make cDNA and then the cDNA was put into a plasma library*
Contains DNA expressed in cell from which mRNA was isolated
Does not contain introns or regulatory regions
Useful for studying types of genes expressed in particular tissue type like brain
this is NOT ALL THE DNA OF HTE CELL, if think about how much dna is in cell, not all of it gets made into mRNA there are the regulatory regions all kinds of stuff never transcribed so if you start with mRNA you are getting much less dna then you would in a genomic library but you are also getting a differnet kind of infromation getting info about which genes are being expressed inside the cell, the main reason people get excited about that is becuse it allows you to compare tissues or organs within teh same species, so if looked at cDNA library that came from all mrna genes being expressed in pacnrease vs genes being expressed in teh brain or skin if you have made genomic libraries from a brain cell, skin cell, etc all have exactly the same dna from same person so wouldn’t tell you anything about different gene types, but if start with mRNA can tell you which dna is actyually being expressed
TRANSGENIC ORGANISMS
Have had genes from other species inserted into their genomes
Viral vectors can be used to insert genes of interest
Can create organisms whose entire genomes create transgenes
Add genes of interest into germ-line cells, which → eggs and sperm
Add genes by injection (for flies) or electroporation (for animal cells)
Electroporation = jolt of electricity opens pores in cell membrane, DNA enters
Environmental applications: e.g., bacteria modified to clean-up oil spills, transgenic orngaims is when they talk about genetically modified food etc**
Agricultural applications: e.g., golden rice, make vitamin A
GMOs = genetically modified organisms. Concern about modified genomes escaping
Transgenic organisms 2
you can put a gene from one organism into another random organism, v famous to put green fluorecent protein a gene from jelly fish as proof of concept put gene for GFP into all these different animals
if put a gene from one species into another can get recipient to express a gene or protein it would not naturally make
- means has its own DNA as well as at least one gene or piece of DNA from another organism* for ex the insulin ex we were talking about is transgenic bacteria so has bacetria’s dna and human dna*
transgenic organism- original one from organism adn another gene from another organism ex. gene for spider silk into genes of goats to produce spier silk in their milk, those are transgenic goats have whole goat genome and then one gene for spider silk - that is what a transgenic organism is, then how did you get that gene for silk into the cells of the goats, how did you get to the point expressing silk protein. You need a delivery system and that is what a vector is, you need a way to get transgene into host organism, viruses can wheazle their way into cells and hijact machinery of reproduction os in creation gf transgenic organisms or biotech strategy to take new gene put on virus, virus is a vector meaning any delivery system to get new gene into cell* using a virus** b/c virus will go into the cell so can hook your gene of interest onto the virus
vector= tool for delivering dna, Rna into the cell*
Electroporation =
= jolt of electricity opens pores in cell membrane, DNA enters; membrane becomes pour so can sneak dna in
about how have new piece of dna how do you get it into a cell
Gene therapy
Goal = to deliver wild-type genes to patients with recessive genetic disorders, so goal is to deliver a good allele to someone with two copies of a bad allele* like tay sachs or some example of a recessive genetic disorder, at a locus they have two bad alleles so goal is to deliver one good allele and hope the individual’s cells will express that good allele and make the protein that wasn’t being made properly*
*conseguinity* fancy word for incest production of offpsring for incest and cousins called a consegunitiy
Supply a “good” allele to compensate for the two mutant alleles the person has
Viral vectors typically are used
-if actual theraputic situation* if have two bad copies for a gene important in a metabolic pathway and you put in and expose them to a good copy of the gene, the good allele and you hope it goes into teh right place and you hope it gets expressed only way to know is if their symptoms go down, can make thsi protein couldnt make before and often protein in question is an enzyme can break down someting they could not break down before* how you would assess whether the gene therapy is working*
tons of metabolic diseases where they have a bad allele for something, some homozgous recessive genetic disorders where tehy actualy have two bad alleles and cannot make a protein say some enzyme that they need for metabolism** idea is if you can make a good allele for them, very easy to do that in a test tube, hard part is to get it into their bodies and get them to express allele, goal of gene therapy is to do that, if someone doesn’t have a good copy of an allele to give them one and try to deliver it to their cells and get their cells to make whatever enzyme the person is not normally able to make* and therefore to cure them of whatever disease; many experiments and efforts for a really long time, the hardest part is getting the gene into the cell and getting it ot be expressed properly** ppl have used viruses as vectors or delivery systems, all kinds of nanoparticles now being used to delivery bits of DNA to cells* for our purpsoes to understand the goal if someone has a disease becuse they do not have a functioning allele for a certain thing you want to give htem a copy of a good allele and get their cells to express the protein
REPORTER GENES AND PROTEINS
Reporter gene is attached to the promoter of the gene of interest
Regulated in the same way as the gene of interest
Often used to study patterns of expression (spatial or temporal)
Reporter gene’s product should be easy to detect
Should not usually be present in the system
Example: green fluorescent protein
Example: luciferase (lights up)
Reporter genes and proteins 2
- This lets you know if gene you care about is in the cell
- so link your gene to green flourescent protein, go in together and if cell lights up with green FP then can assume your gene is being expressed also. Valid assumption you see in excets for MCAT arrticles, expression of reproter gene have high expression of green high expression of your gene to get into the cell***
- if trying to put a gene into a cell, idea is that then if you have your gene that is linked together with luciferase, then it is very obvious if you successfully put them into a new cell becuase the cell will light up so term reporter gene is good, luciferase is the reporter gene tells you whether your experiment worked or not, so this is used as a reporter gene, then if luciferase plus the thing you are trying to put in the cell actually went in the cell it lights up
also green flurescent protein is big reporte,r gene trying to get into the cell went it; the point is that it is very very easy to see if they are there, they are not normally there and you can easily tell if they are
BOTH LIGHTS UP, if gene gets expressed very easy to see that is the goal with a reporter gene
Polymerase chain reaction
DNA replication performed in vitro
Can produce billions of copies of a given sequence
DNA strands are separated at high-temperatures
Requires use of Taq polymerase, which is heat-resistant; special form of DNA polymerase that will not be denatured by heat becuase doing all these hot and cold cycles, makes a complimentary strand
Requires sequence specific DNA primers (not RNA primers, as in vivo)
Primers with more cytosines and guanines bind more successfully (more H bonds)
Step 1: Raise temperature to 95°C
• Denatures the DNA, making it single-stranded and thus open to binding primers
Step 2: Lower temperature to 55°C
• Allows DNA primers to anneal (bind) to specific sequences on exposed strands
Step 3: Raise temperature to 75°C
- Allows for rapid polymerization of new DNA complementary to each of the strands
- Taq polymerase, isolated from thermophilic archaea, extends the DNA from primers
Cycle repeats > 30×, to yield billions of copies of DNA
Polymerase chain reaction 2
what are the best primers?
very very efficient way to go from small DNA sample to huge, exponentialy increasing amount of DNA
exponential increase in the amount of DNA
THE BEST PRIMERS have more G and C** because G and C stick best to DNA becuase they have three hydrogen bonds than two, if designing perfect primer want more G and C vs A and T which form two hydrogen bonds but G and C form 3 hydrogen bonds to complimentary so they are hte best primers*
PCR does not copy….
DOES not copy any markings on the DNA or acetly groups on the DNA
You lose any epigenetic information you are just copying the DNA itself so depending on what people want ot study it may not always be the perfect technique**
electrophoresis
Can separate molecules (DNA, RNA, protein) based on size BIG PICTURE
Run molecules through a sieve-like gel in an electric field
Big molecules move slower, small molecules move quicker
Example: can see change in protein size (e.g., due to nonsense mutation)
For proteins, separation is based on size and charge
Proteins can also be coated in negative charge using sodium dodecyl sulfate (SDS)
With SDS, separation of proteins is based on size ONLY
SDS → breaks weak bonds, denaturing protein. SDS does not break disulfide bonds
Gel run “under reducing conditions” → breaks disulfide bonds
Gel run “under non-reducing conditions” → does not break disulfide bonds
Urea → breaks hydrogen bonds and other weak bonds
Higher or lower pH → disrupts electrostatic interactions
electrophoresis 2
gel, dna negatively charged so have positive end of the gel be below look on image.
Running it through a gel, DNA is negatively charged will move to positive end and bigger pieces of DNA will get slowed down more by the gel, if you run the gel for a certain amount of itme fragments that have gotten further from starting point will be smaller ones so separatign based on size*** TRUE FOR RNA AS WELL!
DNA fragments will move through gel and you get a banding pattern, separating according to size, this is a smaller fragment the other is a larger fragment, and then can think about the DNA having to move through this very thick material so thes smaller pieces can get through more and farther*
Another way to visualize thsi is to blot it onto a surface, you can do a southern blot for DNA
electrophoresis for RNA and protein 3
can do the same thing with RNA called a northern blot, which is still negatively charged
and protein can do it as well, protein can do native page* which is not messing with the protein; so do not denature the protein, and then fragments separate by size and also by charge* whereas DNA and RNA are JUST by size, proteins can be by size and charge
blotting is how you analyze it, meaning you take antibodies that will attack the different specific protein fragments and tell you what you have
Blotting
Southern blot: run DNA through gel, analyze with hybridization
Northern blot: run RNA through gel, analyze with hybridization
Western blot: run protein through gel, analyze with antibodies
analyze with hybridization= other way you can tell, say you wanted to know more about what is in this DNA fragment, you can create a complimentary piece of DNA called a probe and wash it over the gel and see if it sticks* and if it sticks that means it is complimentary to what is on the gel and that means you knwo what sequence is on teh cell, so a way of using probe and technique of hybridization of what is on the gel*
PAGE with SDS
- coats protein with lots of negative charge, and then the separation is JUST based on size, because there is so much negative charge that the amount of charge is just proportional to the size so you are really separating according to size
- if run gel “under reducing conditions” break disulfide bonds
- if run the gel “under nonreducing conditions” means you KEEP disulfide bonds*
so you can run the PAGE with SDS under either condition of reducing or nonreducing, either/or condition** how this really comes up when have proteins that have subunits* so lets just say that for proteins with subunits*
Ex. of proteins separating with subunits
- if the only bonds between subunits are weak bonds, meaning not covalent like hydrogen bonds or hydrophobic interactions then SDS** will break the subunits apart* so SDS will interfere if you think about what could coating something with a ton of negative charge disrupt it /could disrupt everything except for covalent bonds**
- if there are disulfide bonds between the subunits, the subunits will only separate if the gel is run under reducing conditions so have to wait for that language*
- so whether subunits stay together or not ends up determining how many bands appear on the gel and how big they are, so fr an example, there are a lot of examples on problem set want to talk about a couple of them at least
- for an example if you have a homodimer, means 2 subunits that are the same size* and say the whole thing is 60 kDa and say this is the native page and you get a band representing 60 kDa
- SDS PAGE- breaks subunits apart if it is a homodimer and hte two subunits are the same as eachother then they both have to be 30 since they are hte same as eachother you would only get one band, same band for 30 kDa= get two subunits broken apart at 30 on top of each other
example heterodimer
we assume no disulfide bonds, otherwise the SDS would not break hte subunits apart because covalent bonds are not disrupted by SDS
- native page get 60 kDa one 40 kDa and one 20 kDa
- for SDS page, now you get a 40 kDa and a 20 kDa so you would get two separate bands** the homodimer is more tricky becuase exactly on top of eachother so that it looks like one band versus two bands*
Page
polyacrilmide gel electrophoresis an acronym for electrophoresis, just the name of the gel*
DNA sequencing
Chain-termination approach:
DNA samples are added to four test tubes
Each tube contains DNA sequence of interest in single-stranded form
Each tube contains normal A,T, C, G deoxynucleotides, primers, and DNA polymerase
Each tube contains a small amount of a “poisoned” dideoxynucleotide
Dideoxynucleotides = ddATP, ddTTP, ddCTP, ddGTP (a different one in each tube)
Dideoxynucleodies = chain terminators. Lack oxygen needed for sugar-phosphate bond
Termination of chain occurs with low frequency and at random places along the strand
Early termination results in short strands and later termination results in longer strands
The poisoned nucleotides may be labeled radioactively or fluorescently
Labeling → allows detection of DNA to which they are attached
Each tube is analyzed for the lengths of its terminated strands (e.g., on a gel)
Strands are arranged in order by length
Each strand ends with a ddNTP
Sequence read according to the ddNTPs
becuase nucleotides are dideoxy no place to add they end the fragment* get fragments of all different sizes and line them up in size order and you can tell what the end letter was in each case
DNA sequencing 2
DNA sequencing purpose is to figure out sequence of strand shown all the way on the left so they are calling it the template strand of DNA
so your goal is to figure out what the sequence strand of DNA is, so doing all the fragments and making them so can lien up complimentary strands so can figure out sequence of originals trand
centrifugation
Can separate molecules and parts of a cell with a centrifuge
In a centrifuge, denser things towards tube bottom (in pellet)
Less dense things stay in fluid (in supernatant), use this to separate big things like organelles from a cell
*separating based on density* more dense things go down**
plasma=proteins, ions nutrients anything being carried in watery part of blood, a lot of water
SiRNA
Small interfering RNA
Is complementary to specific mRNA → binds and prevents translation, it is an inhibitor of translation
“Scrambled mRNA” does not bind to mRNA
“Scrambled mRNA” serves as a control in experiments involving siRNA
siRNA could be used clinically to prevent overexpression of proteins
RNAi interference, made piece of mRNA and that is used to make protein; you would use siRNA or RNAinterference as a way to stop that** so it stops protein production*
the si RNA in particular is complimentary, so that would be the siRNA cells cannot deal with double stranded rna, if have complimentary piece reduce to cell and it sticks to mRNA and makes it double stranded then you will not get the protein, the cell can’t translate the mRNA when it has a complimentary siRNA piece stuck on; ribosomes apparatus can only deal with single stranded*
use this as a technique or can be used clinically in a situation where the problem is too much protein being made overexpression of a protein* they have definitely done experimetnal passages on the mcat, describe this introduce some siRNA and show you graphs indicating less protein gets made bcause of hte siRNA* but experiments always need to have a control group so the siRNA would be the thing that actually reduces protein production, but scrambled RNA is the control and it DOES NOT reduce protein production*** the idea here is that siRNA works because it is complimentary to the mRNA and really sticks to it, scrambled rna doesnt stick to anything so it is used as a control**
CRISPR-CAS9
Gene-editing technology (“gene surgery”)*** subject of lots of battles, can surgically alter dna much much more precise to go in and alter dna compared to using restriction enzymes you can really target specific area vs. restriction enzymes would work at a restriction site, here can be go customize to go cut at a specific point
Can alter DNA in living cells
Short RNA sequences (Crisprs) targets specific DNA sequences
Guide sequences made of RNA called the crisprs and stick use RNA as a guide to where you want to be in the genome and then Cas 9 binds to DNA and does the cutting
Cas-9 binds to DNA and cuts
Crisp gets you where you want ot be and Cas-9 cuts**
CRISP= guides tell system where to go
Cas-9 scissors to cut**
Hybridization
idea of a probe with dna fragment of a certain sequence becuase represents a mutaiton, maybe fragments from a person dna and they have a mutaiton and you think mutaton would be contained in this fragment you can take a probe which is complimentary DNA or RNA and put it in and if it sticks that tells you what is present on your gel*
Hybridization is annealing (pairing) of complementary nucleic acids
Melting = dissociation of complementary nucleic acid strands
Probe is single stranded DNA or RNA and is radioactively labeled
Probe will bind to complementary sequences
Can detect mutations in genes (e.g., deletion) or find similar DNA in different species
Can be used to search genomic or cDNA libraries for genes of interest
Hybridization 2
ex say have a bunch of DNA from a fetus want to know if DNA has a mutaiton oen or both of hte parents have, so take teh fetal dna and spread it out on a gel and then you use a probe, you know what you are looking for you know what mutated sequence is and want to know if dna present ornt in fetal dna, make a probe single stranded that is complimetnary to the mutated sequence; then you run it over the gel, and then you wash teh gel off if probe finds mutated sequence it sticks to it, probe will be something that lights up you can tell it found the mutated sequence and therefre this fetus has the mutation* but if you do all of this and nothing lights up the probe just washes away means nothing for probe to stick to so sequence is not present**
restriction enzyme image
what does it do? it recognizes a restriction site that is a section of DNA that has a palindromic sequence, like GAATTC right to left GAATTC so it is the same = palindromic!
Gene cloning image
https://image.slidesharecdn.com/cap20biotecnologia-131018103028-phpapp01/95/biotecnologa-7-638.jpg?cb=1382092372
genomic library 2
take dna from chimp store in genomic library, has all the dna of chimp cell can compare it to teh genomic library of a yeast cell which has ALL THE dna of a yeast cell
what percentage is the same between yeast and chimps…. YOU CAN COMPARE THIS* how we can compare the whole genome of the chimp and whole genome of the humans or rabbit and say wow look 97% of the dna is homologous or essentially the same where those #s come from
*KEY STARTING WITH ALL THE DNA IN THE CELL*
cDNA Library 2
take mRNA in a cell–> reverse transcribates–> cDNA
have to get reverse transcriptase to get cDNA that letter means it came from mRNA
great for comparing function /activity of different cells
can be used to compare cells within the same organism* if you wanted to know what is different in terms of protein production of pacreatic cell versus brain cell, you need to use a cDNA library** if took all the dna of a brain cell and all the dna of a pancreatic cell, couldnt see the difference if same organism because all of our cells have the same dna, what makes a brain cell look and act differently from a pancreatic cell is what is actually BEING EXPRSSED** so if you want to get a handle on this you need a cDNA library*
SO IT CAN BE USED TO compare cells WITHIN THE SAME ORGANISM*
gene therapy 2
hard part of this is deliverity it into the cell and getting it ot be expressed*** why it is not more widely used why it has not fully taken off it is very very hard to get the good dna to go to the right place to be expressed without messing up anything in the cell. Sending new dna into teh cell can insert in the wrong way, disrupt somethign receiptient gets cancer all bad thigns can happen, so that is the problem what is the vector that delivers the dna and how do you make sure i tisn’t causing other problems*
ALWAYS THE DELIVERY***
Goal = to deliver wild-type genes to patients with recessive genetic disorders
Supply a “good” allele to compensate for the two mutant alleles the person has
Viral vectors typically are used
To know if gene therapy is working…..
investor- is gene therapy working, are they getting better?
Look at clinical represntation of ptient after a while, is their a change are they getting better?
PCR image
Replication, can make enormous numbers copies of a gene this way
Denature= D
Anneal=A
Extension= E
“Dirty Ass Ethan”
Electrophoresis 4
DNA: smaller molecules go farther, separation is based on the SIZE of the DNA fragment
RNA electrophoresis
smaller molecules go farther, separation is based on size!!!
Protein: Native Page Electrophoresis
Protein is not denatured, it retains its shape and its charge
SO separation is based on size and charge, if more highly charged may affect how quickly it moves*
Protein- SDS 5
There is SO much negative charge everywhere it is not about the charge
- Protein is denatured, covered in negative charge
-separates things based on size ONLY
-breaks apart subunits of a protein (UNLESS they are linked by disulfide bonds*) so like trimer with 3 subunits stuck togehter or dimer 2 subunits, any bonds holding those two things together H bonds, electrostic, as long as it is NOT disfuldie becasue that is a kind of covalent bond.
Leaving aside if no disulfide bonds and you put SDS everywhere coating everyhtign compeltely with negative charge so subunits come apart and are repeling each other at that point so can only separate based on size
ELECTROPHORESIS OF PROTEIN*
Electrophoresis “Running the gel under REDUCING conditions”
BREAK DISFULIDE BONDS
-represent own category because covalent bonds not as easy to break as those other bonds in proteins that contribute to 2 or 3 structure*
What are you doing to teh protein to amke one move further than the other?= this is a kind of electrophoresis, are you creating conditions that break apart subunits of protein on the gel*
S-S disulfide bonds, oxidized form
HS-SH reduced form= bond broken
“nonreducing conditions” doesnt break any bonds, means disulfide bonds are not broken*
urea
Denatures proteins, breaks bonds that are not covalent***
beta-mercaptoethanol or DTT
breaks disulfide bonds, it is reducing***
Hybridization 3
Taking a complimentary piece of DNA or RNA flourescently labeled see whtther it attaches ot anything and lights
- Make a probe complimentary to teh dna of interest*meaning it will stick to what you are looking for in a complimentary way* looking to see if you have a certain gene* it will stick to the complimtnary letters probe will light up in some complientary way it will be fluorescent* if it sticks you can tell because you see the lighting up. There is a spot that is glowing becuase probe stuck to something, I know the sequence of hte probe so I know what it stuck to must be complimentary to that so must have that sequence
After running the gel, how do you where the fragments are? how do you knwo what you have
- hybridization
- blotting
Blotting 2
Banding patter shows up on film with radiaoctivity or shows up becauseof silver chemicals
treating gel in some way to make it visible, can use radioactivity to have film exposed or can do it with silver the poitn is you are making the bands visible*
“autoradiography”
film= literally like photos
siRNA 2
if someone is over expressing a protien, you ptu something in to stick ot mRNA and if you make it double stranded by putting in what is shown in blue as complimentary strand that will block translationa nd you will nto get so much protein* so many therapies make sue o fthis
- very useful if problem is too much protein production*
Blotting mnemonic
SNOW
DROP
Q9. In order to compare promoter sequences used by mice, cats and rabbits, researchers would most likely use:
a. genomic lbraries created from mRNA
b. cDNA libraries created from mRNA
c. genomic libraries created from DNA
d. cDNA libraries created from DNA
c. genomic libraries created from DNA
In order to compare promoter sequences, promoters are not transcribed so there is no mRNA for the promoter sequence, you just get mRNA for the gene itself!
If there is no mRNA then it wouldnt matter about cDNA, cDNA all comes from mRNA so you wouldnt have any mRNA and you also wouldn’t have any cDNA corresdponding to the promoter region, so if you want to study promoter region across species a cDNA library would be completely unhelpful
Promoter sequences are not transcribed SO NO mRNA*** corresponding to promoter, promoter sequences would nto show up in cDNA*
Q10. Which of the following is NOT true of gene therapy?
a. researchers might deliver genes to target cells using a modified form of HIV
b. researchers are likely to explore this approach when a dominant genetic disorder produces overactive proteins
c. Researchers are liekly to explore this approach for recessive, metabolic disorders in which enzymes are rendered non-functional
d. researchers are likely to explore this approach for diseases that do not have other successful, treatments.
b. researchers are likely to explore this approach when a dominant genetic disorder produces overactive proteins
Gene therapy trying to put in a good copy of an allele, the problem is that there is a protein that is missing or mutated. You are not getting a protein that you want to get becuase of a genetic mutation, so gene therapy tries to supply a corrected allele it tries to make up for the fact that there is a gene problem that makes a certain protein not be created.
What gene therapy is really good for=replacing something that is broken but if the problem is that the gene isn’t broken just being massively overproduced gene therapy will not help with that. Gene therapy is supposed to replace something, gene therapy is good for replacing something that is broken or missing it is not good for suppressing protein expression that is not what it does*
looking for something that is not true of gene therapy, when a dominant genetic disorder replaces over active protein issue her is too much protein, gene therapy helps when there is too little protein or no good protein, it does not help when hte problem is too much protein that is the opposite problem*
Q. 20 Which of hte following has the LOWEST electrophoretic mobility upon native PAGE (gel electrophoresis)?
a. 10 kDa monomer
b. 100 kDa monomer
c. 5 kDA homodimer
d. 50 kDA homodimer
b. 100 kDa monomer
we are looking for lowest mobility would be the biggest! sticky adn thick, made of native page so no subunits are broken down anyway so monomer is
page= abbreviation stands for plyacrymide gel electrophoresis, just a type of gel
Q. 21- Which of the following will have the GREATEST electrophoretic mobility in SDS-PAGE udner nonreducing conditions? assume no disulfide bonds are present.
a. 3 kDA monomer
b. 75 kDA monomer
c. 4 kDA homodimer
d. 100 kDA homodimer
c. 4 kDA homodimer
greatest electrophoretic ability in SDS page under nonreducing. All negative charge if coat all subunits negative, actually all repel eachother if two separate things getting negtively charged will break apart. told no disulfide bonds present, can hold subunits geothermal even if SDS around but no disulfide bonds so don’t even have to think about that. we want just hte greatest electrophoretic ability* we are looking for SMALLEST
b is already bigger than a, c the 4 kDA homodimer. The dimer part means two subunits, this means that the subunits are the same size as eachother* so if total has to be 4 and the two subunits have to same size they are 2** if told it is a heterodimer than would not know and can be 3.5 nad .5 we don’t know but with homodimer we know what they are!!! they get separated by SDS
SDS breaks these up into two separate 50 kDA subunits but that is still much bigger 50 and 50 is still much much bigger than 2 so left with c
Q22 - Which of the following will have the GREATEST electrophoreitic mobility in SDS- PAGE under reducing conditions (assume no disulfide bonds are present)
a. 3kDa monomer
b. 75 kDa monomer
c. 4kDa homodimer
d. 100 kDa homodimer
4kDa homodimer
reducing conditions are irrelevant becuase no disulfide bond to break, will be smaller than the 3 kDA* so we want smallest and answer is 4 kDa homodimer
Q23. A heterodimeric protein contains no disulfide bonds. How many bands will it form in native PAGE and in SDS- PAGE under nonreducing conditions respectively?
1 and 2
native page has no reagent that would break apart the subunits* nothing so 1 band stays together totally versus SDS they separte so 2 bands
it is really simple no charge is being added nothing is being done, protein as is is going down the gel* ads page other scenario doing that with the same thing so native page stays together 1 band
when adds sds-page: coats everything in negative charge everywhere so they would repel eachohter* so you get two separate subunits* and importantly they are different from eachother so you would get 2 bands** b/c heterodimeric you would get a band for a and a band for b***
Q24. A homodimeric protein contains no disfulide bonds. How many bands will it form in native PAGE and in SDS-PAGE under non-reducing conditions, respectively?
1 and 1
homodimeric, native page runs down gel 1 band
sds-page they will split up, but because they are the same size as each other will still only have 1 band. Separate but still same size!
Q25. A heterodimeric protein contains two subunits joined by disulfide bonds. How many bands will it ofrm in SDS-PAGE under nonreducing conditions and SDS-PAGE under reducing conditions respecitvely?
so sds page, nonreducing conditions 1 band b/c even though all these negatives it is nonreducing conditions so does not break disulfide bond and still get 1 band.
sds page, reducing conditions: A- SH + HsB means get two bands so the idea is that the SDS page reducing conditions breaks disulfide bonds and that means the subunits separate* and you get 2 bands*** becuase a and B are different sizes*
nonreducing conditions cannot break disulfide bonds* so even with negative charge they are stuck together*
In vitro vs. in vivo
In vitro- inthe lab, in test tube, culture dish, or elsewhere outside a living organism.
In vivo= performed or taking place in a living organism*
siRNA
Page
Native vs. SDS
PAGE= the GEL
2 types of reagent, aka what you put into the gel*
- Native- native no denaturing reagents
protein keeps it s conformation, implies proteins are in their native state they have not been denatured*
- SDS- coats protein with negative charge and is denaturing. Also will cause subunits to separate, unless they are held by disulfide bonds****
when they say sds page means sds in the gel
How does scrambled RNA works as a control?
siRNA works to inhibit translation is complimentary to the mRNA so binds
scrambled is some other peice that is not complimentary and will not bind to the mRNA, think about control group fo an experiment lets say youy have a piece of SiRNA to use theraputetically put into cells to prevent translation of a piece of mRNA to prevent oversexpression of aprotein causing problems in a clinical situation, so siRNA is a way to reduce translation of protein many proteins you want to produce less of
run the experiement and show what is happenign less expression of a messed up protein or something you had too much of, but then the FDA says wait a minute have to narrow it down and show actually that that specific piece of RNA is what was doing the job not something about the way you were experiementig agent put in, or anything put in would have same effector any piece of RNA would have hte same effect, so you with your control experiemnt have ot say now; i am puttign in another piece of RNA and you still get translation** sso that is part of the process of PROVING that your siRNA is really an active ingredient really doing what you are saying doign what you want it to do
that is the logic of having a control* scrambled rna not complemntary, wont bind to mrna to prove you really know what is generating the effect that it really is yoru experimental compound
the end point you are looking at in your study is less production of protein, what if whatever process you are using to put siRNA in is causing damage to not produce protein, no can do an identical process to put something very very similar in cell and I still do not get a protein, not just ht eprocess of adding rna to a cell to give me this effect it is specifically this siRNA drug trying to get approvable*
After recombinant plasmid….
