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Flashcards in Unit 1 Deck (103):
1

DNA stands for

deoxyribose nucleic acid

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bases of DNA

adenine, thymine, guanine, cytosines (T:A and C:G)

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where do the bases attach on DNA?

1 prime carbon

4

T:A bonding is

2 hydrogen

5

C:G bonding is

3 hydrogen

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phosphodiester bond

link nucleotides together (covalent)

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explain phosphodiester bond reaction

reaction between 3' OH on existing strand of DNA and 5' phosphate on free nucleotide

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RNA vs DNA

-ribose vs deoxyribose
-uracil vs thymine
-single stranded vs double

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RNA and DNA

-phosphodiester bonds
-base pairing

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protein structure

-primary
-secondary
-tertiary
-quaternary

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describe primary protein structure

string of amino acids connected by peptide bonds

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describe secondary protein structure

3D shape of the protein due to R group interactions

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describe tertiary protein structure

3D shape of the protein due to R group interactions

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describe quaternary protein structure

when multiple protein monomers interact

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chromosomes

how DNA is packaged inside the cell (nucleus for eukaryotes)

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in a single cell DNA is how long

2m

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nucleus is how wide

0.006 mm

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DNA gets wound tightly around

histone proteins

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histone

protein DNA gets wound around

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nucleosome

histones and DNA wrapped around histones (segment)

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chromatin

DNA and protein being packaged to make chromosome

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chromosome organization

-DNA is always packaged around histones
-tightly packaged DNA is harder to transcribe
-histone protein is made of 8 monomers

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packaging influences

transcription/gene expression

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monomers of histone protein

H2A x2, H2B x2, H3x2, H4x2

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regulation of chromosomes packaging

a. Chromatin remodeling complexes
b. Histone variants
c. Histone modifications

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chromatin remodeling complex

big complex of proteins that binds a nucleosome and moves the histone and expose promoter to start transcription

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histone variants

H2A, H2B, H3, H4

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example of different versions of histone monomers that will change packaging

H3.3 (locked open conformation and can't tightly package)

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monomer

single protein (peptide)

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dimer

2 proteins coming together for a functional unit

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homodimer

2 of the same proteins

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heterodimer

2 different proteins

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histone is a (BLANK)

protein

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protein is a (BLANK)

chain of amino acids

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amino acids are made of

R groups

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examples of histone modifications

methylation, acetylation, phosphorylation, ubiquination

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histone modifications occur

on long tails of histones

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how do the modifications influence packaging

-some modifications make it easier to tightly package the chromatin
-some modification make it more difficult to tightly package the chromatin

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histone modifications can be inherited from one (blank) to the next

generation

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2 mechanisms of epigenetics

1) histone modifications
2) methylation of DNA itself

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it is harder to transcribe (BLANK)

highly methylated DNA

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DNA methyl transferase protein adds methyl groups to new strand of DNA after regulation

CPG methylation

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all enzymes are

proteins but not all proteins are enzymes

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maintaining loosely packaged chromatin

-DNA gets replicated
-histones get divided between 2 new strands
-histone modification on old DNA gets REPLICATED
onto new DNA
-old modification recruits the protein that replicates the modification

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Ethidium bromide experiment

-small fluorescent molecule intercalates into DNA structure
-UV light excites EtBr, visualize the light

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radioactive phosphorus 32P

expose film to the radioactivity

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electrophoresis

separating DNA molecules by size

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gel material for electrophoresis

agarose

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apply an electrical current to gel and (BLANK)

-smalller pieces of DNA move more quickly than larger pieces

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steps to separate and visualize DNA

1) get your DNA
2) separate by size using electrophoresis
3) visualize with EtBr/UV light

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RNA

single stranded

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steps to separate and visualize RNA

1) get RNA sample and denature hairpin structures using fermeldehyde
2) separate using electrophoresis
3) visualize with EtBr/UV

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steps to separate and visualize protein

1) get your protein sample and denature with BME chemical, SDS, or heat
2) electrophoresis
3) visualize (stain)

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amplify DNA and RNA

PCR (polymerase chain reaction)
-make more copies of a specific sequence of DNA
-use PCR primers

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southern blot

DNA detection

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southern blot steps

1) isolate DNA samples of interest
2) separate DNA by electrophorsis on a gel (flimsy)
3) transfer/blot DNA onto a membrane (sturdier)
4) detect your DNA sequence of interest (use of fluorescent probe)
5) visualize where the probe is on the membrane

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probe

piece of DNA that base pairs with DNA sequence of interest

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membrane

film that gets exposed by fluorescence

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application of southern blot

paternity test

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steps for a paternity test

1) isolate DNA
2) cut DNA up into smaller pieces (restriction enzymes cut at specific sequences)
3) isolate by size
4) transfer to membrane
5) probe
6) visualize probe

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restriction enzymes for paternity test

if shared with mom/dad will share restriction enzyme places and will look similar

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probe does what for paternity test

30 bp long
-probe hybridizes in small place on chromosome

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northern blot

RNA detection

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northern blots steps

1) isolate RNA from samples
2) separate RNA by electrophoresis
3) transfer to membrane
4) probe specific for gene of interest
5) visualize probe with film

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northern blot example

which tissues express PECAM-1 gene

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western blot

protein detection

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western blot example

is a protein of interest present?

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steps of a western blot

1) isolate proteins from samples
2) separate proteins (size) by electrophoresis
3) transfer to membrane
4) probe
5) visualize probe with film

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probe for western blot

antibody- proteins made by immune system
-really specific binding interaction (antibody#1 can bind protein A but not protein B)

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loading controls

all cells have similar amounts of GAPDH or actin gene expression

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how to amplify DNA and RNA

polymerase chain reaction

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polymerase

enzyme that replicates DNA

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ingredients for PCR

-DNA to be amplified
-polymerase enzyme
-free nucleotides
-primer

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Why PCR?

need more DNA to visualize or want to visualize a specific sequence

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application of PCR

detect specific DNA sequences (GMOs)

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amplify RNA

reverse transcriptase PCR

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RT PCR steps

1) isolate RNA
2) turn RNA into DNA--> reverse transcriptase (using RNA template to synthesize DNA)
3) PCR

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how to detect interactions between biomolecules

-EMSA
-ChIP
-CoIP

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protein and DNA/RNA assay

EMSA and ChIP

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protein and protein

coIP

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Reverse transcriptase PCR

-RNA sample
-used as template to synthesize DNA
-cDNA (complementary)

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how to visualize results on non quantitative PCR?

-run samples on an agarose gel, visualize with EtBr
-plant DNA

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non quantitative PCR shows

blobs correspond to size of DNA that was amplified (how far up or down on gel corresponds to bp or primer location)

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conclusions we can make from non-quantitative PCR

-is it there? (yes/no)
-how long is it? (bp)

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How to quantitate PCR results

SYBR green fluorescent dye and Taqman probe

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SYBR green fluorescent dye

small molecule intercalates into DNA structure

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qunatitative PCR steps

1) denature
2) anneal
3) extend
4) quantitate SYBR

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what does SYBR tell you

how much DNA you have amplified

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CT value

come up with normalized DNA/mRNA amount

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no normalized mRNA means

fluorescence never crossed threshold and no cDNA template)

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EMSA

interactions between protein and DNA

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EMSA stands for

electromobility shift assay

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DNA and protein can be (BLANK) with EMSA results

heavier or slower than DNA alone

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steps of EMSA

1) amplify DNA seq of interest
2) get your protein of interest
3) incubate DNA sequence and protein
4) run a gel and visualize DNA

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ChIP

chromatin immunoprecipitation assay

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ChIP question

is a certain protein binding to a certain sequence of DNA/RNA

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steps for a ChIP

1) crosslink the binding between DNA/RNA and protein
2) isolate DNA/RNA and protein to break into smaller pieces
3) immunoprecipitation of protein and DNA/RNA complexes
4) determine what DNA sequences were immunoprecipitated (pulled down)

98

questions to ask at end of ChIP

-did my DNA sequence of interest get pulled down?
-purify DNA that got pulled down and then do PCR with primers that will amplify DNA seq of interest

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immunoprecipitation

-use an antibody (+ bead) specific against protein of interest
-spin in a centrifuge
-antibody + protein/DNA will be at the bottom

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CoIP

coimmunoprecipitation

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goal of CoIP

looking at proteins that bind to each other so could be immunoprecipitated together

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question while doing CoIP

does protein Y bind to protein Z

103

steps of CoIP

1) get your proteins- isolating from a cell or use purified proteins
2) incubate your proteins + antibody specific against a protein of interest
3) spin the tube down in centrifuge
4) do western blot on pulled down proteins (pellet)