Bio 401 Exam 3 Notes

3/11 Cloning
Goals
 to play with genomic DNA to see what it does
 Take alcohol of some sort and detergent and some strawberries to see the DNA
 Need to find the sequence with shot gun sequence or whole genome sequence and then
amplifying it with PCR or something
om
Recombinant DNA
 1. Extract genomic DNA from donor organism - we don’t clone at the level of RNA, we clone at
r.c
level of DNA.
 2. Identification of gene locus - do this by knowing a sequence and identifying regions to make
lur
primers to amplify regions.
 3. Cloning of gene into vector molecule (plasmid, cosmid, virus) - find some sort of DNA we can
stick DNA into
nb
 4. Introduction of insert/vector into host cell to replicate (bacteria, yeast). Yields millions of
copies of insert (clones) - doesn’t matter what species we get our DNA from, we can use whatever
tu
system we want. Ex. Work on humans and find important gene and you want to make millions of
copies. You would amplify it and put it into bacterial vectors. Doesn’t matter that it's bacterial bc
ar
DNA is DNA
 5. Verify identity of insert by mini/midi/maxi- prep
Sm
 Get your DNA, have a vector, place the DNA into the vector, place the vector into the cell, and it
will replicate
via
e d
ar
Sh

is
file
is
Th
Miniprep
We provide unlocked studymaterials from popular websites at affordable price, email enquiries to rishabhk28@live.com

om
r.c
lur
nb
tu
ar
Sm
via
e d
ar
Sh

is
file
is
Th
 Vector with our DNA in it. Take the DNA and place it into the tube with vectors. Hope the DNA
ligates into it. Place it into a pool of bacterial cells. Hope that our construct gets incorporated into
the bacterial cells. Get multiple colonies after the cells replicate
 Recover the plasmid and make sure your DNA was incorporated. There's going to be genomic
DNA and lipids that you don’t want. All you want from a miniprep is to get rid of everything else
and just have your DNA. Need to verify that what you have is your DNA.
Cloning
 Allows the amplification and recovery of a specific DNA segment from large, complex DNA
om
sample.
 Utilizes chimeric (human DNA into bacterial) DNA and simple cell systems
o Ex. Human DNA/ bacterial vector
r.c
lur
nb
tu
ar
Sm

via
e d
ar
Sh
is
o Bacterial cells have circular chromosome but they also have little circular pieces of DNA
called plasmids. They're small circular DNA and have antibiotic resistance genes (and if it
file
conjugates with another cell, it can give the resistance to another).

o Need to cut it open to have bases that will H bond with each other. Cleave with
restriction enzyme which leaves overhangs and if you cut your foreign DNA with the same
is
enzyme, it will have complementary DNA and can ligate together.

Th
om
r.c

lur
nb
tu
ar
Sm
o Transformation - getting foreign Dna into a cell. Cells don’t just pick up DNA - they need
via
to be capable or you need to treat them chemically. In this case, they have competent cells
that are willing to take up DNA and then you have to change the temperature or something
that way it can pick it up.
d
DNA Isolation
e
 First step of cloning

ar
 Isolate donor and vector DNA

Sh
 Majority of donor DNA will be nuclear

 Can use:
o phenol/chloroform
is
o DNAzol®
o Commercial DNA extraction kit
file
Cutting DNA
 Use restriction enzymes
is
 Recognize and cut within or outside specific sequences

 Named after the bacteria from which the enzyme is isolated
Th
o Ex. EcoR1
 Isolated from E. coli
 Cuts GAATTC - cuts between the GA to create overhangs
 Know EcoR1, HindIII, Sma I, BamI
 Can cut blunt ends or sticky ends
o Sticky ends - have overhangs. One strand is going tqwo be longer to have free
nucleotides
o Blunt ends - no overhangs. Cuts straight down
 Depending on ends, can be used for directional or non-directional cloning
o Sticky end is used for directional cloning. Sometimes you want to insert DNA in a
particular orientation. If there are overhangs, you can ensure your DNA is in the correct
om
orientation and isn't upside down or backwards
 Can recognize different number of bases (ex. 4-cutters vs 6-cutters)
o When working in the lab and you know the sequence, you need to use the right
r.c
enzymes to make sure it clones. Cant always use the same enzyme
 Ave. distance between cuts depends on # of bases the RE recognizes
lur
o Make a somewhat accurate prediction how often the enzyme will cut in a sequence of
DNA. If we use a 6 cutter instead of a 4 cutter, just because of a random chance, 6 cutters are
going to cut less often that 4 cutter.
nb
o 4n= ave. distance between cuts- n= # of bases recognized by enzyme
 At any given site, you can any 4 bases.
tu
 4-cutter- 1 cut each 256 bp
 6-cutter- 1 cut each 4096 bp
ar
 Going to be variation based on richness of certain nucleotides
Sm
 Why does it matter? If you have a million bases, it's better to cut them into
bigger pieces to work with. Different vectors hold different sizes
via
e d
ar
Sh
is
file
is
Th
om
r.c
lur

nb
tu
ar
Sm
via
e d
Cloning of DNA
ar
 Vectors for cloning

Sh
 Will hold the inserted DNA from donor

 Must contain:
o 1. Origin of replication for host cell - trying to insert a DNA and make millions of copies
is
so you need site where replication can start

o 2. Multiple cloning region (or polylinker) - sequence along the vector that's going to have
file
many restriction enzyme sites of different enzyme. If you have to cut with BamH1 and
there's no BamH1 cutting place in the vector, you can't insert the DNA
o 3. Resistance gene - allow them to survive. Agar plate with minimal media, stuff can
is
grow on the plate so anything that grows into a colony, it can be anything. What we normally
do is place an antibiotic so anything that grows has a resistance gene. So anything that grows
Th
has the resistance

o 4. Marker gene - lies with multiple cloning region in the middle part. There will be a
promoter that turns the gene on. It might have the B galactosidase gene as a marker a to
whether or not there is an insert. If you took the plasmid without the insert and stuck it into
a plate with lactose substituent and if it gets cleaves it forms a blue precipitate. If you cut
open the Z gene and interupt the operon with our own DNA, we will not produce functional
B galactosidase so it will not be blue. We look for blue vs white colonies
om

r.c
lur
nb
 There are a lot of overlapping colonies. That is not good. It is difficult to
pick a colony and only pick one. You want to spread them out better so the
tu
colonies are spread out from each other.
ar
Sm
via
e d
ar
Sh

is
file
is
Th
o Worry about the one on the right.
o There are 4 things the plasmid needs. There needs to be an origin of replication to
replicate. You cant put one of a human into a bacterial cell bc it wont recognize the region -
needs to be recognized by the host cell. The plasmid lies off the main chromosome and
allows it to have antibody resistance. Stick is back into a bacterial cell with ampicillin. There
is a site with restriction enzyme recognition sequences where the plasmid will be cut. This
om
lies in the middle of a lacZ gene. There are two steps to finding the cells that we want. The
first step is see which cells have the sequence. If the plasmid doesn’t have the insert, it wont
grow on the ampicillin plate (called selection). There are cells with plasmids that can still
r.c
survive. If the plasmid doesn’t have the insert, the lacZ will be active and it will create B
galactosidase. On the plate, there is something similar to lactose and if there is B
galactosidase, it will react with it and create blue. If it has the insert, it disrupts the lacZ gene
lur
and the color will be light.
nb
Types of Vectors
tu
1. Plasmid- Bacterial in origin. Small, circular DNAs that lie off the main
chromosome.
ar
 Replicate independently of the bacterial chromosome - important bc it doesn’t need to wait for
Sm
the bacterial cell to divide. You can get cells with multiple plasmids
 Can hold up to 20 kb of DNA but they themselves are at the most 5000 base pairs long. - can't
stick really long pieces of DNA. Most people only clone up to 1000 or 2000
 Ex. TOPO®. Don’t worry about the top part. One of the interesting things about Taqp is it puts an
via
A on every 3'. Theres a cloning kit that uses this vector and it has T overhangs so anything made
with Taqp will let it ligate easily.
e d
ar
Sh
is
file
is
Th
om
r.c
lur
nb
o
tu
ar
Sm
via
e d
ar
Sh
2. Phage lambda
 Virus particle - form plaques. E coli will cover the entire plate because they will want to eat the
virus. The virus will insert its dna into the bacterial cell and the plaques mean that the bacterial
is
cells have been blown up.

file
is
Th

 Can package a chromosome up to 50 kb
 Cut out central part of vial chromosome with restriction enzymes- only “arms” necessary for
replication and packaging - take viral genome and cut out the middle part and leave the arms
which allows them to go through the litic cycle.
om
r.c
lur
nb
tu
ar

Sm
via
e d
ar
Sh
o Cut DNA into small sequences. They did the partial digestion where you don’t expose
the DNA to the restriction enzyme for the entire time. Letting it go for the entire time allows
it all to be cut but this restricts the time and doesn’t cut at every single site but just some
is
sites so you'll have different sizes. Get rid of the yellow part and you can stick it into the
middle of the virus as long as it's not at one of the arms.
file
o Make individual viruses with a right arm an a left arm and whatever we stick in between.
We're growing ecoli and allowing the viruses to attack the bacteria.
Expression Vectors
is
 Contain enough info for transcription and translation of insert in bacterial cell
Th
 Inserts must be stripped on introns

 Human gene into a bacterial system and put under control of a dysome system. This gene will be
expressed.
 Need to get rid of all the introns and use just the cDNA. The bacterial cell wouldn't know what to
do with the mRNA and introns. It would just read right across. So you need to start at the level of
the mRNA and then do reverse transcription to get cDNA and put that into the cell.
om

r.c
lur
nb
o T7 - site for polymerase
tu
o If you put the insert in backwards
o 5' at the correct site and 3' at the correct site along with the correct frame
ar
o They put their gene of interest under transcription factors that are only expressed in
specific cells to find out where it's being expressed
Sm
Yeast Artificial Chromosomes
 Put back into yeast cells
 Can hold up to 1,000,000 base pairs
via
 Very large pieces of dna (millions of base pairs) - has to have centromere, origins of replication
Human Artificial Chromosome
 Make an artificial chromosome w centromere, telomeres, and origin of replication
d
 Lie in the cell in the nucleus as an extra chromosome

e
 Bare essentials to act as a chromosome but problem of gene dosage doesn't come into play
ar
Bacterial Artificial Chromosomes

 Bigger than a plasmid
Sh
DNA libraries
 Collections of cloned DNA fragments that are packaged together
is
 Two types
o 1. Genomic Library- A collection of DNA clones that contains at least one copy of every
file
sequence in the entire genome

 Take genomic DNA and cut it up but what you're trying to do is have at least one
copy of every single sequence in the library
is
 Made up of everything - protein coding, introns, promoters, etc

Th
o 2. cDNA Library- Collection of cDNA clones that correspond only to those genes being
expressed at a particular time
 Not all the sequences that are found but the genes that are being expressed at
that moment
 Starts with the extraction of mRNA and RT to get cDNA and insert into phage
particle
o Liver cells and kidney cells would have the same genomic library but different cDNnA.
om
r.c
o
lur
nb
tu
ar
Sm
via
o
e d
ar
Sh
 DNA is all the DNA. cDNA is based on what's expressed

Screening the library
is
file
is
Th
om
r.c
lur

nb
tu
ar
Sm
via
d
 Take cellular genome and cut it with restriction enzymes and ligate and insert into the
e
recombinant DNA to produce a different phage particle. There will be a bacterial lawn grown on a
ar
plate and they will inject their own DNA onto the cell. End up with plaque. Green is bacterial cells
and black is the viruses that are left there.
Sh
 Take a special paper called nitrocellulose paper

 Each plaque corresponds to one particular virus.
is
DNA Binding Assays Lecture 1

Immunoprecipation
file
 Uses an antibody (-immuno) attached to a bead to purify a protein

o Agarose, sepharose, magnetic
 Similar to an affinity chromatography
is
 Using antibodies that is tethered to some sort of bead whether it's magnetic or agarose and that
allows the protein to be pulled out of the solution because those beads are insoluble.
Th
 Take your bead tethered to an antibody and you need to place them into a cell lysis solution. You
take your cell and lyse them and take out all the protein that is available. Then your antibody can
bind to your protein. You then take the test tube and spin it down so that the beads can propel to
the bottom of the test tube. You can take off the remaining protein that didn't bind. That gives you
just the precipitate at the bottom which is your protein and your antibody. You can blot a few
times to get rid of any protein that didn't bind to your antibody or protein. At that point, you
should end up with just the protein and antibody so that you can reverse the antibody binding
reaction to get just your protein. This is to enrich or purify your protein.
 You could use magnetic beads as well and then put a magnet on the side and then centrifuge it.
Streptavidin magnetic beads binds to biotin - important molecular interaction and strongest
interaction we know and use. Have a biotinylated antibody which would bind to the bead and
you'd be able to pull the protein out.
om

r.c
lur
nb
tu
ar
o Protein a/g acts as an intermediate between the bead and the antibody
Sm
via
e d
ar
Sh
is
file
is
Th
om
r.c
lur
nb

tu
ar
Sm
via
e d
ar
Sh
is

file
is
Th
Advantages
 Allows purification or enrichment of protein of interest from a solution that contains a lot of
proteins
 Many different variations - look at different types of interactions
Disadvantages
 IgG remains in sample – will overlap on SDS-PAGE with proteins of 25 & 50 kDa
o If you were to western blot it
o Try to find proteins that are not 25 and 50 kDa
o If you were to immunoblot it, you wouldn't be protecting your antibody at all you'd be
looking at your protein of interest so that's not that big of a disadvantage
 Can pull out other proteins that nonspecifically interact with bead, protein A/G, or antibody
om
o Monclonal antibody only binds to one part of the protein. Polyclonal binds to several
different parts of the protein. If you use a monoclonal, you're more likely to get specific
reactions. There are some very sticky proteins out there. There are proteins you can pull out
r.c
without intending to. Never going to be exactly what it is
lur
Types
 Co-IP – Protein-protein interactions. Pulling out your protein plus any proteins that are bound to
nb
it
o SH2 and SH3 domains - generally cause protein protein interactions. If your protein has
this, you can do a co-IP to see which other proteins it binds to.
tu
 ChIP – Protein-DNA interactions
ar
o ChIP-Seq – determine DNA binding sequence. Pull out the DNA and sequence the DNA.
o ChIP-chip – determine relative DNA binding affinity. Once you pull out the protein and
Sm
the DNA, you can run the DNA over a DNA microarray and compare relative binding affinities
for two different conditions, like cancer vs non cancer cells
o ChIP-exo – degrade extraneous DNA to only get exact binding sequence. While the
protein is still bound, you can treat the DNA with exonuclease. This starts at the end of the
via
DNA and starts cutting the nucleotides until they can't anymore because it's blocked by the
protein. This narrows it down to the exact sequence that we want to the binding sequence.
o A lot of the times you do chip you pull out large fragments of DNA where you don't
d
know where the DNA is going to bind to

e
 RIP – Protein-RNA interactions (ribosomes)

o RIP-chip – determine relative RNA binding affinity. Pull out RNA, reverse transcribe the
ar
RNA, and use a microarray to compare relative binding affinity.

Sh
o CLIP-Seq – Determine RNA binding sequence. Crosslinking immunoprecipitation. A lot of

the times RNA-p interactions are not well bound so you can use radiation like UV light to
crosslink them together and this should help with pulling them out. Then you sequence it to
get the RNA sequence.
is
o PAR-CLIP – Use photoactivatable ribonucleosides to enhance crosslinking. CLIP is

relatively ineffiecient at crosslinking. Use particular ribonucleosides that get incorporated
file
into the RNA and they can be induced to improve crosslinking effieciency at particular
wavelengths. Enhancing CLIP
is
Fast Protein Liquid Chromatography FPLC

Th
 Used to purify proteins and enrich them

 Nickel column - create protein in some sort of vector. When you do the pcr process before
attaching your protein to your vector, you're going to attach about 6 codons that encode for
histidine at the end, usually the C terminal (instead of the N - interferes with the protein more).
Histidine has an imidazole side chain that will bind to nickel. Clone the protein with the 6 histidines
under an inducible promoter - some type of molecule that you can add to your bacterial growth
medium that will cause this particular protein to be transcribed. So then you have tons of proteins
in your sample and so then you can lyse the cell and extract the protein that will put the protein
lysin on the nickel column and the imidozole group of the 6 histidines will interact with the nickel
and causes binding. Everything that doesn't have histidine will run through the column. Then you
can dilute your protein of interest by adding imidizole and increasing gradient into the column. As
you add this, you start to displace the amount of histidines attached to the column since it's not
om
strictly imidizole and just a side chain. At different concentrations of imidizole, you can collect
different fractions and then you can run a western blot to see which fraction your protein ends up
on.
r.c
Co-immunoprecipitation (Co-IP)
lur
 Method to observe protein-protein interactions in vivo or in situ.
Should be in the same conformation they should be in natively - they should have any
nb
o
type of post-translational modifications. In vitro you might not have the same (advantage of
co-ip)
tu
 Same principle as IP - pulling down proteins that are going to be bound to your proteins
Uses:
ar

o Different treatment conditions - breast cancer cells with each treated differently. Look at
Sm
how the interactions change based on the treatment.
o Time course - at 0 hours what is the protein interacting with? 4 hours? How do they
change over the course of treatment?
o Different cell types - liver cells vs kidney cells. just because they have the same protein
via
produced doesn't mean it's going to bind to the same proteins

o Domain specific interactions - you can go in and mutate a particular domain and see if
your protein still binds to a protein. If it doesn't, that would be indicitive that the protein
d
binds to that protein at that domain.

e

ar
Sh
is
file
o Little bead attached to the antibody with a complex of proteins that gets pulled out. You
can reverse the binding reaction and a gel to separate all your proteins and you can then
is
immunoblot them to see which ones are there if you know which ones you're looking for,
Th
and if you don't you can cut the bands out and digest them and submit them and get them
identified.
Advantages
 In vivo / in situ
 Proteins are in their natural conformation
 Proteins are likely already post-translationally modified
Disadvantages
 Misses weak interactions - sometimes there're things that happen really quickly or loosely bound
proteins or proteins that are associated with each other but don't really interact and it'll miss
these
 May be a complex of interacting proteins - it can pull out your protein, the protein it's attached
to, and the protein that that one is attached to. You can't really say that your protein is interacting
om
with the last one since there is one in the middle - all it tells you is that it's a complex
 Need appropriate antibody
 Lots of steps to mess up
r.c
False Positives
 Appears positive from the results of your experiment but isn't actually there. Something that
lur
shows you there is an interaction that isn't happening
 Proteins do not directly interact but interact in complex
nb
 Nonspecific antibody binding or bead binding - see them on your western blot or gel but it's not
actually interacting with the protein
tu
 Compartmentalization - when you lyse the cells, you're spilling everything out. There could be
proteins that are separated by compartments but when you spill them all out, they could interact
ar
with each other that wouldn't necessarily be interacting with each other. There are ways to get
around that. There are some solutions that only lyse the cytoplasm membrane and some that only
Sm
lyse the nuclear membrane.
False Negatives
 Proteins that do interact but don't come up in the results
via
 Weak interactions - could get washed away in all the steps

 Circumstantial interaction - a lot of the results are a snapshot and show what's happening at that
exact time. Some proteins may interact but didn't happen to be interacting when you conducted
the test.
d
 Antigen binding site is blocked - the antibody could block another protein from binding
e
ar
Yeast2Hybrid
Sh
 Also looks at P-P interactions and usually for global screenings

o Done before co-ip - see all the proteins it's interacting with. Unless you have a particular
target, you generally start here and then use co-ip to confirm
False positives - done globally
is
o
 Using a yeast system but usually looking at proteins that are not yeast proteins. When you
produce those proteins in yeast, they might not fold the same way or have the same post
file
translational modification occurring.

 Two vectors. Target protein and a bait protein. Clone these into the two vectors. Gal4
transcriptional activator has an activation domain and a DNA binding domain. These two domains
is
need to be together in order for transcription to occur. You're creating two different proteins and
Th
on one protein you're going to use your activation domain to your bait protein on your one vector,
and on the other you're using your DNA binding domain to your target protein. What you're going
to end up with is your target and your bait that have an activation domain and a binding domain
tethered to them. This can create problems. When you attach a domain to a protein that wouldn't
usually be there, the protein may not fold correctly. What happens is, if your two proteins do
interact, that will cause your activation domain and your DNA binding domains to come into close
proximity. They can then bind to the Gal4 promoter and promote transcription. Generally there's a
reporter gene like lacZ and if your proteins do interact, you’d have transcription of lacZ which
would cleave xgal (similar to lactose) and create a blue precipitant.
 Blue = interaction, white = no interaction
 Might be blocking an interaction by adding on another domain
om
r.c
lur
nb
tu
ar
Sm
via
e d
ar
Sh
is
file
is
Th

om
r.c
lur
nb
tu
ar
Sm
via
e d
ar
Sh
is
file
is
Th
Chromatin Immunoprecipitation (ChIP)
 Identify recognition sequences of DNA-binding proteins in vivo or in sit
o See what exactly the protein is binding to in terms of promoters, enhancers, and
silencers
 Native ChIP vs. Crosslink ChIP
o Native - not producing a crosslinking reaction - doing what is natively going on in the cell.
Not always great bc you can miss a lot of interactions by just washing away the DNA from the
om
protein
o Crosslink - POI to the DNA
 Process:
r.c
o Crosslink – formaldehyde or UV. Protein is not going to let go of the DNA despite the
washing steps
lur
o Lyse cells
o Sonicate/RE digest to shear/fragment DNA - breaking up the DNA into small pieces. You
don't want to precipitate a whole chromosome. Sonication uses sound waves to break them.
nb
o Immunoprecipate - antibodies to pull out your protein and the DNA binding protein
o Digest protein/purify DNA
tu
o Analyze DNA
 Targeted - if you know what genes you're looking at
ar
 Genome wide Sm
via
e d
o
ar
Sh
is
file
Targeted
 Southern blot - ran out the DNA on the gel and probe for the gene you're looking for.
is
 Semi-quantitative PCR
Th
 qPCR
 Have to know which DNA sequence you're looking for.
Genome Wide
 Cloning – create library. Take the pieces of DNA and clone them into a library and then you can
work from there for further analysis
 ChIP-on-chip/ ChIP-chip - pull down the protein with the DNA and then do a DNA microarray to
look at binding affinity.
 ChIP-Seq - figure out which sequence you're binding to
 ChIP-exo - take exonuclease and chew up until he hits the protein and that would just leave the
sequence your protein is binding to
om
r.c
lur
nb
tu
ar
Sm
via
Advantages
d
 In vivo / in situ
High throughput
e

Disadvantages
ar
 Lots of steps to make mistakes

Sh
 Extensive optimization required - lots of troubleshooting to find the best conditions to do

something in
 Need specific controls - if you don’t have controls, you can't validate your results
is
 Need very specific antibody

 Sometimes requires known target sequence - genome screening you don’t need it
file
 Antigen must be accessible -if that part of your protein is bound to the DNA the antibody will not
be able to bind
False Positives
is
 Transient DNA binding - sometimes the protein isn't bound to the sequence but stays near the
sequence. If you crosslink, you could get a protein that's near that site but doesn't actually interact
Th
with it
 Nonspecific antibody binding - pick up some other type of protein
False Negatives
 DNA binding can be very circumstantial - based on what humidity and temperature
 Inaccessible antigen
RIP/CLIP Advantages & Disadvantages
 Just know that CLIP is inefficient at crosslinking and PARCLIP is used to enhance it
Electrophoretic Mobility Shift Assay (EMSA)

 Verifies Protein-DNA binding in vitro
om
o Completely in vitro - happens in a test tube
 This is what you use to validate CHIP
 DNA is labeled - looking at DNA not protein
r.c
o 32P
o Biotin - binds to strepdavidin
lur
o Digoxigenin - molecule that is specific to plant and is used in animal models so that it
doesn't bind to anything
o Fluorescent - fluoroflore
nb
 Have a binding reaction. Take your labeled DNA and put it in the testtube and you take your
protein or your cell lysin and put that in the test tube too. You let them incubate in the test tube
tu
for about 30 minutes or so at which point you take that solution and run it through a gel. The DNA,
which is the smallest, will run to the opposite side of the gel. If your protein binds to the DNA, it
ar
will produce a larger complex which will mean it will not run as far. It will shift the band up in the
gel.
Sm
 Looking at the DNA - seeing the DNA binded to the protein not actually seeing the protein
 If protein binds to labeled DNA creates larger complex – shifts band UP in gel
via
e d
ar
Sh
is

file
is
Th
 Treated the breast cancer cells with doxorubison and 5-florouracil which are two
chemotherapeutic treatments used. Both of these are mutagenic and p53 responds to breaks in
DNA and both of these cause the DNA to be mutated which causes the upregulation of p53. In A,
we have normalized the amount of protein added not specific to p53. When you normalize the p53
in B, the binding drops in the treated one. The treated one causes a spike in p53 but the actual
binding decreases. It's not specifically binding to those genes it is just there in general which is why
om
there's a larger intensity for the untreated vs the treated.
 "p53 responds to DNA damage. When you damage DNA, you get an uppeak in p53. p53 binds to
the p21 promoter which upregulates p21 and the cell cycle stops. P53 targets genes that are
r.c
involved in dna repair. If it can repair, the cell cycle starts again. If it cannot, it promotes apotosis. if
you put the same amount of total protein in each cell, p53 would be a larger percentage in treated
cells than nontreated cells. The shift in band intensity stays the same across. If it is a larger
lur
percentage, wouldn’t you expect it to be more intense? If it has the same DNA binding affinty, you
would expect more binding. The untreated has a higher band intensity – p53 has a higher affinity
nb
to bind in untreated. BINDING AFFINITY DECREASES WITH TREATMENT WITH THE DRUGS. It drops
but there’s so much there that it keeps binding. A – same amount of protein. B - same amount of
tu
p53."
ar
Sm
via

e d
ar
Sh
is
file
is
Th
om

r.c
lur
nb
tu
ar
EMSA Controls Sm

via
e d
ar
Sh
is
file
is
Th
 Always need the first lane of th eleft - DNA labeled only. Need to know where the DNA runs
when it's by itself so you know if there will be a shift or not. Second lane does not have DNA at all
and it's only protein. Need to do this so that you know you’re not detecting your protein. Have a
DNA + Protein lane so you can see your shifted band if there is one. You need to make sure your
protein is binding to that specific DNA sequence and not just DNA in general - there are some
proteins that will bind to DNA regardless of the sequence. There are other proteins that only bind
to specific sequence. To make sure it's not random, you have to add a random piece of DNA which
is the 4th row. Non sensitive DNA to make sure the band doesn't change and stays the same as
protein + DNA. Need to have a specific competitor to make sure that the protein is specifically
om
binding to that sequence. Youre going to take your labeled DNA and generate it without the label
and add it in in excess (1x labeled, 100x unlabeled). That should outcompete the labeled DNA if
the protein binds to both to the same affinity. This way, the band will be there but it will be part of
r.c
the unlabeled DNA. This makes sure that it binds to that specific sequence. The last lane is protein
+ DNA, + antibody to your protein. This results in a larger complex and this creates a supershift.
You may want to check that by using a nonspecific antibody that would not shift the complex up.
lur
RNA EMSA
nb

tu
ar
Sm
via
e d
ar
Sh
is
 Exactly the same thing except labeled RNA instead of DNA

file
Streptavidin Magnetic Pull-down Assay (Reverse EMSA)

 Use biotinylated DNA - DNA template has biotin which binds to strepdavidin
 Insert protein and it binds together
is
 Have protein-DNA binding reaction

Pull out protein-DNA complex using magnetic beads
Th

 SDS-PAGE + Western blot to probe for protein
 Looking at the protein - not the DNA
o Opposite of EMSA

om
r.c
lur
nb
tu
Sequencing Lecture 2
ar
Definition of a species? Two organisms can breed together to reproduce viable organisms. Except
Sm
bacteria don't reproduce. Look at similarities in 16s rRNA which is part of the small subunit of the
prokaroyote in the ribosome. This needs to function well in all organisms for an organism to survive
therefore similar species will have very similar RNAs.
via
BLAST
 Basic Local Alignment Search Tool
o Formed by NCBI, NIH, and NLM
d
 blastn – nucleotide BLAST

e
 blastp – protein BLAST

ar
 blastx – Search protein database using translated nucleotide query

o Takes strand and reads it in all 6 reading frames and then translates them all and
Sh
searches the database for anything that matches them

 tblastn – Search translated nucleotide database using protein query
o Reverse translates and brings it back and reads it in all 6 reading frames
is
 tblastx – Search translated nucleotide database using translated nucleotide query

file
Alignments
 Global
o Take a sequence and compare it to a sequence that is already there
is
o Whole protein sequence – assumes proteins are similar over entire length & evolved
Th
from same ancestral molecule

o Useful for 16S rRNA alignment & proteins that are similar over entire length
 Local
o ONLY regions that have HIGH sequence similarity
o Have an entire genome sequence and you're looking for one gene - it will find that one
gene. Only comparing what aligns with each other
 Based on:
o AA similarity & sequence similarity
 Have charged and noncharged amino acids and look at the similar properties to
see if it would affect the folding
o Evolution
 Frequency of AA substitutions in proteins from different species
om
 Comparison to ancestor
BLAST Results
r.c
 Query - what you imputed
 Subject - what is already there
lur
Scoring
nb
 Look for alignments with
o High levels of sequence identity
tu
o Few mismatches
o Few gaps
ar
o Little opportunity for improvement Sm
 Substitution matrix values are assigned based on frequency of that kind of substitution in a given
collection of protein sequences
o Substitution - penalty
o Insertion - penalty
via
o Match idenity - positive score
PAM and BLOSUM

d
 PAM – Percent Accepted Mutation

e
o Works on entire protein sequences

ar
o Older so based on FEWER protein sequences

o Adjust the matrix for stringency
Sh
 1 for similar one, 250 for different ones, 120 for nice medium
 BLOSUM – Block Substituted Matrix
o Works on blocks where related protein sequences tend to be the same
is
o Newer so based on MORE protein sequences

o 180 - compare similar ones
file
 Both PAM and BLOSUM substitution matrices are based on sequences of related proteins, and so
reflect frequency of substitutions that have not been selected against through evolutionary time

is
Th
Results Scores

om
++ - exact match to your score
r.c
o
 Bit score – takes raw alignment score and normalizes it to scoring system
o Higher is better match
lur
 E-value (Expect) - The number of different sequences where the alignments would yield scores
equivalent to or better than S just by chance
nb
o Lower is more significant score
 Identities – Number of exact matched residues in alignment
tu
 Gaps – number of gaps in alignment
 Strand – same strand or complementary strand
ar
EXPASy
Sm
 Swiss Institute of Bioinformatics (SIB) Bioinformatics Resource Portal which provides access to
scientific databases and software tools (i.e., resources) in different areas of life sciences including
proteomics, genomics, phylogeny, systems biology, population genetics, transcriptomics etc.
via
 Can use to find ORFs - http://web.expasy.org/translate/

 Translates ALL 6 reading frames – Look for longest ORF
d
ClustalW
e
 Open in Biology Workbench (http://workbench.sdsc.edu)

ar
o Username = Molgen16
o Password = Genetics
Sh
 Multiple sequence alignment

 (+) is a conserved residue
 Can use multiple sequence alignments to create phylogenetic trees
is
 Select unrooted tree & all default settings

 Import Alignment
file
Unrooted & Rooted Phylogenetic Trees

 Unrooted tree – NO ancestral root
is
o Use alignment tools in biology workbench

Th
o DRAWTREE – default settings

 Rooted tree – reflect most basal ancestor of tree
o Use alignment tools in biology workbench
o DRAWGRAM – default settings
 Bootstrapping – Assesses reliability of partitions (nodes) in a given tree
o Use CLUSTALTREE in biology workbench alignment tools
o Change BOOTSTRAP OPTIONS
 COMPUTE: TreeONLY à COMPUTE BootstrapTree
o HIGHER Bootstrap value = More confidence in node
4/4 Cancer
om
r.c
lur
nb

tu
ar
Sm
via
e d
ar

Sh
is
file
o Cancer cells produce survival cues and don’t listen to death signals – uncontrolled
is
survival
Th
o Changes at the level of transcription, post transcriptional modification, translation, and

post translational modification
o With cancer, all controls are bypassed
Cell Cycle
om
r.c

lur
nb
tu
ar
Sm
o M – mitosis
o G1 – make sure that the cell is ready for replication. If there are mutations that are
recognized by the enzymes that scan our DNA, they have to be fixed because if mutation
via
occurs they will be maintained

o S-replication
o G2- starts the whole process of getting ready to divide
d
o Cyclins – proteins that dimerize with a partner and find a target to phosphorylate. They
e
heterodimerize with Cdk. A Cdk is a generalist kinase (finds a substrate and phosphorylates)
ar
– it’ll phosphorylate anything you put in front of it. * The cyclins find the target and Cdk
phosphorylate – cyclins tell the cdk what to phosphorylate. *
Sh
o Midway in G1 phase – activation of cdk 4 and cyclin d. Retinablastoma (RB) releases E2F
(transcription factor) which finds enhancer sites for proteins that are necessary for
replication. It goes into the nucleus and transcribe dna polymerase 1, pcna (- forms a
heterotrimer with 3 of itself and become the sliding clamp), and rnr. Without it being
is
released by RB, none of these will be produced. As long as e2f is connected to RB, it stops
the cell before replication. If there's a mutation, it starts a pathway for the Rb to hold on to
file
the e2f. Cyclin d targets RB and cdk4 phosphorylates on a couple of residues. This changes
the conformation of the protein. Once this is done, they become downregulated. Cyclin e
and cdk2 then become activated. These hyperphosphorylate RB and changes its
is
conformation so that it releases e2f. This can become a major issue in cancer cells. If there is
Th
a way for the cell to always bypass the controls, it will keep dividing.
o G2 phase – cyclin b and cdc2 (or cdk1) – become active with a dephosphorylation.
Becomes active with cdc25 phosphotase – removes a phosphate to make them activated.
o G0 – differentiation. Apotosis.
Nomenclature
 Tumors are classified according to tissue of origin
 Carcinomas from epithelial cells
 Leukemias and lymphomas from blood cell precursors
 Major problem:
o Every tumor is individual. Within a family, the mutation may be the same but in order to
get to a tumor level, there need to be multiple mutations for multiple genes so everyone is
om
different.
o No single treatment exists to cure all cancers. Some cancers are better treated with
some forms of medicines than others and some people respond better to certain treatments
r.c
than others.
lur
Cancer
 Cancer occurs when cells continuously proliferate
nb
 Due to mutations in cell cycle regulatory genes
 Different genes that may be mutated
tu
o Signal genes - control the cyclins
o Proliferation genes
ar
o Cell cycle checkpoint genes - cyclin d, cyclin e
 5-10% due to familial genetics Sm
 Rarely, if ever, caused by a single mutation
 Usually the result of an accumulation of mutations during the proliferation of somatic cells
 Metastasis - transformation from benign tumor to a tumor where cells break off and reestablish
themselves in other places is the problem
via
 Ex. Colon tumors contain mutations in 5-10 genes

 Average of six to seven successive mutations needed to convert a normal epithelial cell into a
malignant, invasive carcinoma
d
 Can have 100’s of mutations in cell before change to cancerous

e
 Made up of cells (clones) descended from one cell

ar
 Mainly result from exposure to mutagens, not through the germ line
 Not only are we still being bombarded by carcinogens, but since they’re replicating so quickly,
Sh
they are probably making more mistakes and mutations.

 Rates of cancer incidence varies due to location of population
o Environmental cues dictate what type of cancers are seen.
is
o However, immigrants begin to show the same cancer rates as indigenous people
 Tumors usually develop over time
file
o Ex. Lung cancer incidence increased two decades after men began frequent smoking
o Incidence of cancer rises with age
o Suggests that cancer-causing mutations accumulate over time.
is
Tumor Growth
Th
 Mutations found in tumors are one of two types

 Those that improperly activate genes
o Ex. Proliferation genes
 Those that improperly inactivate genes
o Ex. Cell cycle inhibitors
o Inactivating the cell cycle inhibitors can be a problem as well
 Cki – bind to cyclin to prevent them from functioning.
o Two main groups – p16 (INKS) and the p21 family (WAF).
o Prevents the complex from phosphorylating RB. This keeps the e2f so it cannot get into
the nucleus so there's no TF formed.
o P53 – TF that is activated in response to DNA damage and when it becomes active, it
om
turns on the CKI genes.
Cancer Genes
r.c
 Oncogenes- Mutant alleles that act dominantly to promote proliferation. Can be heterozygous
for the condition
lur
o .Cyclin d1 is a very well known oncogene. With a mutation, it becomes active
 Proto-oncogene- non-mutant precursor to oncogenes.
nb
 Mutant tumor-suppressor genes- Mutant alleles that are recessive. Both copies must be mutant
to make the cell abnormal
One good copy is still going to do what it needs to do. Act in a recessive way so that both
tu
o
copies have the effect
ar
Sm
via
e d
ar
Sh
is
file
is
Th

Bio 401 Exam 3 Notes

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Bio 401 Exam 3 Notes

Uploaded by

Copyright:

Available Formats

3/11 Cloning

conjugates with another cell, it can give the resistance to another).

enzyme, it will have complementary DNA and can ligate together.

 First step of cloning

 Isolate donor and vector DNA

 Majority of donor DNA will be nuclear

 Recognize and cut within or outside specific sequences

 Vectors for cloning

 Will hold the inserted DNA from donor

so you need site where replication can start

has the resistance

cells have been blown up.

 Inserts must be stripped on introns

 Lie in the cell in the nucleus as an extra chromosome

Bacterial Artificial Chromosomes

sequence in the entire genome

 Made up of everything - protein coding, introns, promoters, etc

 DNA is all the DNA. cDNA is based on what's expressed

 Take a special paper called nitrocellulose paper

DNA Binding Assays Lecture 1

 Uses an antibody (-immuno) attached to a bead to purify a protein

know where the DNA is going to bind to

 RIP – Protein-RNA interactions (ribosomes)

RNA, and use a microarray to compare relative binding affinity.

o CLIP-Seq – Determine RNA binding sequence. Crosslinking immunoprecipitation. A lot of

o PAR-CLIP – Use photoactivatable ribonucleosides to enhance crosslinking. CLIP is

Fast Protein Liquid Chromatography FPLC

 Used to purify proteins and enrich them

produced doesn't mean it's going to bind to the same proteins

binds to that protein at that domain.

 Weak interactions - could get washed away in all the steps

 Also looks at P-P interactions and usually for global screenings

translational modification occurring.

Chromatin Immunoprecipitation (ChIP)

 Lots of steps to make mistakes

 Extensive optimization required - lots of troubleshooting to find the best conditions to do

 Need very specific antibody

Electrophoretic Mobility Shift Assay (EMSA)

 Exactly the same thing except labeled RNA instead of DNA

Streptavidin Magnetic Pull-down Assay (Reverse EMSA)

 Have protein-DNA binding reaction

 blastn – nucleotide BLAST

 blastp – protein BLAST

 blastx – Search protein database using translated nucleotide query

searches the database for anything that matches them

 tblastx – Search translated nucleotide database using translated nucleotide query

from same ancestral molecule

o Match idenity - positive score

PAM and BLOSUM

 PAM – Percent Accepted Mutation

o Works on entire protein sequences

o Older so based on FEWER protein sequences

o Newer so based on MORE protein sequences

 Can use to find ORFs - http://web.expasy.org/translate/

 Open in Biology Workbench (http://workbench.sdsc.edu)

 Multiple sequence alignment

 Select unrooted tree & all default settings

Unrooted & Rooted Phylogenetic Trees

o Use alignment tools in biology workbench

o DRAWTREE – default settings

o Changes at the level of transcription, post transcriptional modification, translation, and

occurs they will be maintained

 Ex. Colon tumors contain mutations in 5-10 genes