Assays and Techniques

Overall purpose of GWAS and QTLs

statistical methods to identify regions of the genome that influence biological traits in a population (link a genotype to a certain phenotype)

QTL molecular question

What specific regions of DNA are responsible for the phenotypic variation that we’re seeing surrounding this particular quantitative trait?

General overview of how QTLs are preformed?

Pick your parental lines (two parents that are genetically distinct for the trait of interest), cross the parents over and over again until you develop progeny families like F2 populations, recombinant inbred lines, or double haploids, phenotype and genotype every individual in the crosses, then generate a linkage map based on recombination frequencies (use statistical analysis to correlate the genotype data with the phenotype data)

what do the results of QTLs tell you?

identify genomic regions that are linked to a certain trait, usually as some kind of LOD score (so a peak above a certain threshold that indicates a high probability that a QTL exists at that position in the genome)

Molecular question of a GWAS

which common variants across an entire genome are associated with a specific phenotype or disease?

How to do a GWAS

Pick your cohort, genotype, association testing, quality control, look for any associations (usually in the form of a manhattan plot- each dot is a SNP, looking for ones above a certain threshold)

what do the results of a GWAS tell you?

It identifies genomic loci that are associated with a trait which can help figure out new candidiate genes and biological mechanisms that are associated with a disease or phenotype

Big thing to keep in mind for GWAS results tho

ASSOCIATION DOESN”T EQUAL CAUSATION, so usually a GWAS identifies a tag snp that is in linkage disequilibrium with the actual causal mutation

What do epigenetic assays/ techniques let you study?

the epigenome which is made up of chemical modifications to DNA and proteins that regulate gene expression but don’t actually change the genetic code itself

Question behind Bisulfite sequencing (epigenetics)

What specific bases in the genome are methylated? Is the promoter of this gene of interest getting silenced by really high levels of 5-methylcytosine?

How do we do bisulfite sequencing?

1) Dna extraction, 2) bisulfite conversion- chemically treating ss dna with sodium bisulfite whichs converts UNMETHYLATED cytosine bases into uracil SO THE METHYLATED CYTOSINES STAY AS C’s 3) PCR amplification- so the converted uracils get amplified as thymines, so methylated cytosines are C’s and un methylated ones are T’s 4) sequencing and compared the sequences to the original reference genome to figure out which cytosines weren’t methylated

what do the results of bisulfite sequencing tell us?

let you make a dna methylation status map across an entire genome (high methylation in promoter regions usully means gene silencing is happneing because chromatin stays really tightly wound, while low methylation means that gene expression is actively happening)

ChIP-Seq (epigenetics)

chromatin immunoprecipitation sequencing

question behind chip seq

Where does a protein of interesed like a transcription factor or mutated protein bind to genomic dna? What are the histone modification patterns that are associated with a specific region in the genome?

How do we do chip seq?

1) Cross link and fragmentation- proteins get chemically crosslinked to the dna they are binding to and chromatin gets fragmented, 2) immunoprecipitation- so an antibody thats specific to the target protein gets used to pull down the protein-dna complex, 3) purification- the crosslinks get reversed and the dna gets purified, 4) sequencing- the fragments get next gen sequenced and mapped back to a ref genome

what do the results of chip seq tell us?

Genomic loci that show up as peaks where our protein of interest could bind, lets us identify the regulatory targets of proteins and map epigenetic markers

ATAC- seq (epigentics)

Assay for transposase- accessible chromatin

what us da question behind atac seq

what regions of the genome are in an open or accesible state (aka euchromatin) vs what regions are in a closed state (heterochromatin)?

How might we preform ataq seq?

1) cell prep- so hrvest our cells, wash em, and lyse to isolate just the nuclei, 2) tagmentation- incubate nuclei with Tn5 transposase to cut ACCESSIBLE dna and ligate sequencing adaptors, 3) purification- purify the transposed dna to remove transposase and debris, 4) library amplificcation (PCR amplify purified library to add barcodes), 5) clean up, 6) sequencing

what do ataq seq results tell us?

peaks that represent regions where Tn5 transposase inserts sequencing adators which menas that there is open chromatin there, and these accessible regions usualy have active promoters, enhancers, and regulatory elemtns where transcriptional machniery binds

BIG THING TO NOTE IN ATAQ SEQ RESULTS

ONLY TELLS YOU IF THERE ARE ACCESSIBLE REGIONS OR NOT DOESN’T TELL YOU IF ITS BEING TRANSCRIBED THO

CHART (epigenetics)

Capture Hybridization Analysis of RNA targets

question that we ask when doing CHART

Where does this long non coding RNA bind in the genome? What proteins are part of the complex it forms at these genomic locations?

How do we do CHART

1) crosslink and fragmentation, 2)hybridization- short biotinylated dna oligonucleotides complementary to the incRna of interest get designed (1-3 probes) and the probes get incubated with the fragmented chromatin at a certain temp so they can hybridize, 3) Immobilization- complexes are captured using streptavidin (SAV) beads and then rinsed to get off any non specific/ non bound chromatin, 4) elution and analysis- the targeted material is eluted from the beads and then can be analyzed with qPCR or with CHARt seq deep sequencing to see any genome wide targets

what do chart results tell us?

gives us a functional analysys of Inc RNAs (or long RNAs) by mapping their genomic occupancy and indentifying what protein partners they recruit to modify chromatin states or modify gene expression (so basically the precise genome-wide, IN VIVO and IN CELLULO mapping of where long non coding rnas bind to chromatin

key tools in gene editing and transgenics

molecular vectors used to deliver genetic material into cells

plasmid vectors (gene editing and transgenics) question

how can we get a gene into a host cell in order to use it to produce our protein in a controlled way or to amplify our gene of interest?

what does making plasmid (circular dna) vectors require?

recombinant cloning- inserting a gene of interest into circular dna

how can we make and utilize plasmid vectors?

1) make ur insert- so you need to isolate and amplify the specific transgene or gene of insert using PCR (within that there is TA cloning so when u use taq polymerase for pcr, the enzyme usually adds in a deoxyadenosine 3’ overhand to the ends of the fragment), also note that ir ur doing a gibson assembly- if more than one fragment is being inserteed, primers get designed with homologous overhands that match the vector, 2) select ur vector 3) ligation- the insert and vector need to get joined together, 4) transoffrmation- so putting the recombinant plasmid into a host cell which is usually e.coli via electroporation (using electric feilds to make temporary pores in the cell membrane so the DNA can enter) or chemical permibilization so resuspending cells in calcium chloride followed by a heat shock, 5) screning and ocnfirmtaion (antibiotic selection, collony pcr, blue/white screening)

what components need to be included in a vector in order for it to be functional?

there needs to be an origin of replication, MCS (multiple cloning site- short region that has unique restriction enzyme recognition sites where foreign dna can be inserted, selectable marker/ reporter gene- usually an antibiotic resistance gene or visible marker like gfp to know which cells have taken up your plasmid, inducible promoter- system like the lac operon or tet on/pff could be put in there too to allow for temportal control over gene expression

what do the results of doing a transformation with plasmid vectors tell us?

that you got successfull expression of the transgene which then lets you preform functional studies of the protein or the production of theraputic agents

Bacterial Artificial chromosomes (BACs)

follows a specific workflow that lets it handle a much bigger amount of dna compared to regular plasmid vectors (derived from e.coli f plasmid)

question behind BACs

How can we clone and mintain incredibly big fragments of genomic DNA (100-300 kb) that include regulatory elements too?

How to create a BAC

vector and insert prep (bacterial f plasmid gets linearized- cut with restriction enzymes at lacZ gene), ligation, transformation, blue/white screening

results of BACs

stable maintenance of a huge genomic interval which is needed to sequence the human genome and identify distant regulatory elements

Viral Vectors

engineered to hijack natural viral infection mechanisms in order to get a genetic cargo into cells

Adeno- associated viral vectors question

How can we deliver a small genetic load for LONG TERM expression while trying to minimize an immune response?

when to use adeno associated viral vectors

human gene therapy, preferably for non dividing cells

how shall we make adeno associated viral vectors

construct design, transfer plasmid, packaging plasmid, helper plasmid, transfection, extraction and purification, titration (to measure the concentration of functional vral particles before its ued in vivo)

results of using Adeno associated viral vectors

the linear AAV dna gets processed into a double stranded circular episome that stays in non dividing cells for years without permanent chromosomal integration

adeno viral vectors question

How can we deliver a big and complex genetic package that exceeds adeno associated vectors?

when to use adeno viral vectors

when the transgene is big (can support loads up to 8x adeno associated ones can)

results of using adeno viral vectors

high efficiency delivery of big genetic packages BUT they do tend to illicit a stronger immune reponse

lentiviral and retroviral vectors question

how can we permanently integrate a transgene into the genome of a dividing cell

when to use lentiviral or retroviral vectors?

to target dividing cells or to do a long term gene knockdown using shRNA

How to use lenti or retro viral vectors?

the vectors can deliver RNA cargo that gets converted into DNA and integrates directly into the chromosome of the cell that ur targeting

results of using lenti or retro viral vectors

permanent genetic modification of the host cell line which allows the transgene to be passed on to allllll its daughter cells

Microinjection

technique used to deliver genetic material right inro a cell (used mostly to create transgenic mouse lines)

What do the results of microinjection tell you?

• Integration Success: The results confirm whether the foreign DNA has been integrated into the host genome, which can occur randomly through Non-Homologous End Joining (NHEJ) or precisely at a target location via Homologous Recombination.

• Phenotypic Impact: By observing the transgenic animal, researchers can determine the biological function of the introduced gene and its role in disease pathogenesis or normal development.

• Expression Patterns: If a fluorescent reporter like GFP was used, the results visualize exactly where and when a gene is active within the tissues of the living animal.

• Production Capacity: For medical applications, the results demonstrate whether the animal can successfully produce functional human proteins that can be harvested for pharmaceutical use

gene knockdown

technique used to reduce the expression of a target gene or the amount of protein produced at the RNA level

what kind of effect do knockdowns have?

Transient

why do a knockdown and not a knockout?

to study genes that might cause the organism to die it they were completely knockout but you still want to know the function of them

CRISPERi/ cas9 interference (gene knockdown)

variation of crispr/cas9 system specifically for a knockdown, which lets reseachers have a targeted repression in transcription of a certain gene without changing the dna sequence permanently

so is crispri still acting like a pair of scissors?

Nope, instead it acts like a physical roadblock to transcription (like a body in the road)

componenets of CRISPRi

1) dead cas9- inactive version of cas9 so that it can’t cut/ make ds breaks, 2) repressor domain- ex KRAB, helps to silence target gene, 3) single guide RNA- the rna molecule that guides the cas9 repressor to the specific 23 bp target sequence

steps of crispiri

1) figure out what gene you want to target, 2) make the guide rna that’ll hybridize to the target DNA sequence, 3) make the complex so let the cas9 KRAB complex bind to sgRNA- sgRNA leads complex to a target spot right next to a PAM sequence, 4) transcriptional intereference- the cas9 represor binds and physically blocks rna pol, boom gene is knowcked down (can’t be transcribed)

results of CRISPRi

effects of silencing transcription of a gene- lets us observe phenotypic effects from losing that gene

Antisense oligonucleotides (ASOs)

short single stranded sequences of DNA or RNA that also knockdown gene function by blocking the expression of ur target gene (hybridize to complementary mRNA sequence)

how to use ASOs

1) design your sequence (its complementary to the mRNA of interest), 2) chemically modify them so that they won’t get degraded, 3) deliver them into the cell (usually gymnotic delivery or lipid transfection), 4) knockdown in the cell

mechanism of action of ASOs

RNase-H dependedt degredation- so ASO binds to mRNA and RNase H recognizes it and hydrolyzes the strand, steric blocking- physcially inhbiting translation, splicing modulation- binds to pre-mRNA to alter splicing patterns (used to exclude soecific exons that might eb associated with a disease)

when do ASO and RNAi interefernce take place in the centrak dogma?

post transcriptionally

results of ASOs

transient knockout

RNAi

hijacking a natural cellular mechanism to use to for a knockdown (reducing gene expression at RNA level) cuz the prescene of dsRNA in a cell gets targeted for degredation

process of RNAi

1) delivery, 2) DICER celavage (like molecular scissors- slices RNA into small pieces), 3) formation of RISC complex, 4) mRNA targeting and Degredatipn (RISC guided by small rna strand, finds taget and binds to coplementary mRNA sequence, then wither degrades it or blocks from translation

siRNA

exogenously produced, shoter transient effect

shRNA

vector based, the host cell makes it (transcribes it) and then it folds into a hairpin structure, longer lasting effect

Inducible promoters

used in knockdown experiments to give a temporal control over gene expression (can turn silencing on or off)

most commonly used inducible systems

tet-on/ tet-off, lac operon

Tet- on/ Tet-off

from e.coli tetracycline resistance operon, uses doxycycline or tetracycle as inducing agent

Lac operon for gene knockdown

lac repressor binds to lac operator to block gene expression, transcription is then induced by adding IPTG (inactivates repressor)

results of inducible promoters in gene knockdown

phenotypic impact of knockdown is measured, BUT inducible knockdowns aren’t instantaneous- there is a delay between adding in the inducer and then seeing an observable reduction in protein levels

Protein protein interactions

studying how proteins function inside a cellular network, BUT keep in mind the choice of method really depends on if the interaction needs to be looked at in vivo so live cells or in vitro and understanding if the interaction is directly between 2 proteins or part of a much bigger complex

Yeast 2 hybrid question

do these two proteins physcially interact with each other? WHat unknown proteins are interacting with my protein of interest?

How shall we do a Y2H

splitting a transcription factor into two parts- dna binding domain (DB) and activation domain (AD), make a fusion plasmid with each domain, the bait protein gets fused to DB and prey protein gets fused to AD. Then transform the constructs into yeast cells that have a synethtic promoter upstream of a reporter gene (usually lac)

results of y2H

if bait and prey interact, Ad gets close to DB and the trasncription factor is now functional so the reporter gene gets turned on and can be transcribed

example of a positiev result in y2h using lacz

the yeast colonies will turn the colonies blue in presence of x-gal

co-immunoprecipitation

to test for direct and indirect interactions of 2 proetins in vivo using an antibody to pull down a bait protein from a lysate

co-ip question

are these two target proteins physically bound to each other in their natural state?

How do we do co-ip?

lyse cells, have an antibody specific to bait protein in lysate making a protein-antibody complex, then have immune complex capture protein, wash the complex to get rid of any unbound proteins, then elute

results of co-ip

analyze elutants via western blot, to see if the prey protein gets pulled down too with the bait

big thing to note about co-ip

it preserves the natural environment of the proteins so it can identify protein complexes so positive result could indicate a direct interaction or indirect interaction mediated by other proteins

Pull down assays

in vitro affinity assay to see direct physical interactions between a known bait protein and suspected or prey protein

how do we do a pull down

tag the bait (GST, HIS, biotin) immobilize it on affinity resin to capture prey proteins that bind to it and separate them from the rest of the lysate

pull down question

can these two purified proteins interact with each other directly outside of a cellular environment (so in a test tube)?

importnat difference between co-ip and pull down methods?

co-ip uses lysate and its done in vivo, pull downs use purified proteins and you do em in vitro

results of pull down

if the prey protein is present in the final sample it confirms that there was a physical interaction

Forster Resonance Energy Transfer

detects PPIs by measuring the energy transfer between two fluorophores (donor and acceptor) attached to target proteins

question of FRET

where and when do these two proteins interact in a live cell?

how do we do FRET?

donor flourophore gets fused to one protein, and acceptor flourophore gets fused to the other, when the donor is excited by light it absorbs energy and emits flourescence, so then you transfect the constrcts into target cells and you need to minter expression levels

results of fret

if the two proteins do interact then the donor transfers energy to the acceptor which makes the acceptor emit lgith, and the donor’s signal deceases, lets us have spatial and temporal resiltion, tagging intercations in real time in living cells But just keep in mind that the flourescet tags can sometimes alter protein function

MOAC

Metal oxide affinity chromatography

question for MOAC

is this protein being phosphorylated?

Protein Assays and Quantification

ways to quantitatively measure the total protein in a sample

when to use UV absorption at 280nm

when you have a known purified protein sample and you know the spectra, and u wanna ask what is the concentration of this specific purified protein?

How to do UV ABSORPTION at 280nm

Assay is based on the beer lambert law (absorbance is equal to extinction coefficent x path length + concentration)

results of uv absoprion at 280nm

direct measurement of the concentration of a pure sample, relies on amino acids so result is depdent on proteins composition

Bradford Assay question

what is the total protein concentration in this detergent free mixed sample?

how do we do a bradford assay?

use coomassie brilliant blue dye to target basic and aromatic amino acids (arginine) causing a color change from brown/red to blue, measured at abrosbance of 595 nm, then us estandard curve to compare unknown readings

results of bradford assay

gives you a sensitivity range of 1-1000 ug/ml (but just keep in minf that proteins need to be <3kDa for this assay)

BCA Assay question

use this when ur sample contains detergents, what is the toal protein concentration in this sample that contains surfactants?