Lecture #8 (Methods) Flashcards
Ways to amplify DNA
- PCR
- Using Bacteria
PCR
Purpose - Amplify DNA
Process - Denature the DNA with heats (dsDNA –> ssDNA) –> Aneal primers (Have primers facing each other annealed/hybridized to a DNA sequences) –> Polymerase will fill in between those primers –> get full dsDNA Strands –> Repeat process (Separate strands again)
- Next round there will be twice as many products (1 –> 2 –> 4 –> 8)
DNA amplification with bacteria
Purpose – Amplify DNA + purify DNA
- Know have pure DNA because the DNA came from one colony that all came from one cell with one plasmid
Process - Cut gene of interest from chromosome –> Add DNA into a plasmid/vector (Use ligase to seal) –> Add plasmid to bacteria –> Bacteria
- Plasmid must have Ori site + Termination = needed for amplification in clone of bacteria cels
Image - hows DNA amplification with bacteria (left) ; PCR on the right
Sanger Sequencing (Overall)
Overall – Use low concetration of dideoxyNTPs (ddNTPs)
Process: Start of reaction – Primer binds to the template –> polymerase begins to add nucleotides (polymerase will mostly add dNTPs) –> eventually polymerase will add 1 ddNTP –> once ddNTP is incorporated the reaction stops
- Reaction stops because 3’ OH is needed to attack the alpha phosphate of the new dNTP = no 3’ OH = can’t continue extending the chain
END – get many fragments with varying lengths
ddNTPs
Chain terminating analog
In Sanger each ddnTNP will have a flourescnent tag
- Flourescent tag is joined by a linker to the base
- DNA polymerase doesn’t care about the flourscent group = is able to incorpoarte the anolog
- If have a floursent tag = can add all 3 ddNTPs in 1 tube
lacks OH on 2’ and 3’ carbons
Anylyzing Fragments from Sanger sequencing
Once have fragments –> denature sample with heat –> run fragments on a poly acrylimide gel –> Gel seperates the fragments by length
As running fragments through the gel a laser at the bottom mesures the flourscence of the fragment –> Can distiguish what the last nucleotide in the fragment is
To determine the sequence - see the peaks of colors for each nucleotide
- Know the sequence of nucleotides by reading the pattern of flouresnece peaks coming off of the gel
Constraint of Sanger Sequencing
Need primer –> means you need to know enough about the DNA to syntehsize a primer
How to sequence DNA if you don’t know enough about DNA BUT need a primer
Purpose - Allows you to sequence unknown DNA
Overall - Clone segments into a plasmid
Put the unknown sequence next to a known sequence in the plasmid vector –> use a primer that binds to the known sequences in the plasmid –> Use PCR to amplify unknown material with primers that binds to the 5’ end of known material on the known sequence at the 5’ end
- Known sequence on the 5’ end = landing pad for the primer
Illumina Sequencing (overall)
Overall – PCR on a solid surface with one DNA molecule seeding each cluster
- DNA sequencing on a solid surface –> image surface after sequential cycles –> different flourscnet signals indicate the nucleotide sequence
Purpose - sequence DNA
- Goal - sequence hundreds of millons of sequences simultenously by atttaching DNA to a surface (glass slide) and look a each point on the slide
- Each point on slide has different sequences = each spot as different flourescnet colors –> miscropce visualizes the colors
Illumina vs. Sanger
Illumina is difefrent from sanger because fixes the issue of having to seperate the product on a gel (Hard to scale up to millions of sequences when using a gel)
Illumina Process
- Build cluster of 10,000 copies of the same DNA sequence fixed on a glass slide (ALL clustered at one location)
- Clustering - Want many copies of 1 DNA sequence in the same place in a cluster on a slide (each cluster has a different sequence)
- After clusters – Add primer in solution –> Primers hybridzie to the end of each strand that is NOT attached to the slide
- DNA polymerases uses the primer for DNA synthesis - Once primers bind add ONLY ddNTPs
- only 1 nucleotide is added each cycle
- ddNTP is added –> color off incoporated ddNTP is imaged by a microscope
- color tells yo what ddNTP is added to that sequence on that spot on the slide
- Once color is imaged - bond between the floruscent tag and the 3’OH is broken – floursecent group is washed away –> 3’OH is revealed –> new based can be added
- Microscope reads the flourescence as each ddNTP is incorpoarted
IN image – Blue bound to the red = primer ; three strands are three different clusters
How do you get clusters in Illumina
Overall - PCR (bridge amilification)
- Start with a peice of DNA that has adapters liagted to the ends (5’ and 3’ end sequences wil hybdrize to the primers on the glass slide ; unknown sequence will be between the oligios) AND have 5’ end of primers are linked to the slide (Blue and red ‘lawn’ on slide)
- 3’ end of the primer is facing up
- Blue adapter on the DNA being sequences will bind to the blue oligio on the slide
- DNA on the sldie is now a template for DNA synthesis –> Synthesize will occur up to the end -> Once synthesise of the complenetray strand is done the 5’ end linkes to the other oligio on the slide (product of synetshize will hydrize to the red oligio if started bound to teh blue) –> NOW that new strand can serve as a template for the next round of synthesis
- End of the fragments = tether to the sldies = product of synthesis can’t be released
END – Glass slide is filled with cluster of DNA with the same sequences that can’t move
Image – Blue binds to the blue on the slide –> DNA folds down and atatches to teh Red –> NOW bridge is the template starting at the red end –> DNA polymerase will sythesize DNA
How do you get clusters of DNA on slide (abridged version)
Condensed version – Once the blue end binds to the slide –> DNA will bend to atatched to red primer –> Bridge will serive as a template to synthesize a new strand
ddNTPs in Ilumina
ddNTP floruscnet dyes are joined to the 3’OH of the ddTNP= block the ability of the 3’OH to attack the next base
- Each ddNTP has different flourescent colors
Imaging Illumina
Microscope images 100 million reactions simultensouly
Image - See microscpe image at different times (At top circle micorscope images green then blue then red – reads DNA seqeuce for the DNA at that spot on teh slide)
Speed of Illumina
Illumina = slow process
Add all 4 nucleotides –> Once cycle of recation (one nucleotide is added) –> Stop reaction –> wash away unrecated based for imaging –> Microscope images –> cleave dye from incorporated nucleotide –> wash away the cleaved tag –> Add reagents again –> repeat
Trancriptomics
Sequencing RNA (technically sequencing cDNA)
Application of Trancriptomics
Transcriptomics = discovery technology (sequence everything)
Example - Have diseased pateints vs healthy pateints –> want to know the difference between controls and dieseaed
- Cluster the data to see how similar the samples are to each other
Example of Trancriptomics data
Top lines = shows the degree of the order of the degree of similarites betwee the samples
- Shows the alzeihermers pateince are more similar to each other than the control
Side – Genes
- Phylogeny lines = shows the genes clustering based on the degree of similarity
Colors shows gene expression
- Shows the genes that are more highly epxressed in controls compared to AD
Example uses of trancriptomics
- Starting point for many experiments –> AFTER can explore the function of the genes
- Detection technology (Diagnosis)
- Can test for the type of AD a pateint has (see if the pattern of expression matches a pattern of expression for a type of AD )
- Test Tumors –> informs decisons in clinic
- Compare the transcriptomes of different tumors
- Might know that one transcriptome pattern predicts responsivness to a drug = can use the transcriptme to decide what drug to give
Single cell sequencing
Overall – Sequencing individual cells
- Uses Microfluidics device
Microfluidics device generting single cells
Overall - Water forms droplets in oil
In Microfluidics device – oil flows in side chanel from the top down and from bottom up AND water flows through middle chanel from left and right –> flow makes H-bond in water causes water to form droplets
- Can encapsulate things in the droplets
Image - Circles in image = droplets of water
Single cell sequencing Process
Uses beads that unique sequence of oligios attatched
- All beads have a different oligios (oligios have a polyT region)
- Beads ALSO contain dNTPs + transcriptase
- Beads = also have a barcode
Process:
Beads flow through a stream of water –> THEN a second stram of aqueous solution with indvoidual disscoiated cells OR individual nuclei merges with stream of beads-> Creates stream of beads and cells –> As stream moves towards right of tube the aquous solution is seoerated into droplets that contain a single cell and a single bead –> lysed cells eleases the RNA from the cell –> PolyT region on the oligio on the bead binds mRNA –> can seperate only the mRNA + PolyT on oligios act as a primer
THEN syntehsize cDNA from the mRNA in the droplet –> Amplify the cDNA with PCR –> Sequence cDNA linked the bead
- Sequencing data will include the barcode AND the unknown cDNA (mRNA) sequence
Beads on in single cell DNA sequencing
Beads have:
1. oligios that also have a polyT region
2. Beads ALSO contain dNTPs + transcriptase
3. Beads = also have a barcode
- Barcode = uniuqe 10-20 nucleotodes that distiguishes THAT bead from all other beads)
- Unique nucleotides = allows reseracher to know that each cDNA came from the same bead
Result of single cell Sequencing
Once you seqeunce PCR product using Next gene –> Get reads for all of the cells
- Each droplet = has 1 cell = each cell has its own barcode (10,000 cels = 10,000 barcodes)
- Can seerpate the reads based on barcodes = know all of the reads that came from one cell
Anylyzing single cell data
Count the amount of reads for each gene
When ploting each gene –> plot for that cell (for 1 barcode) how may read you got
- Example - Gene 1 = 15 reads = point 15 in dimmersnion 1
- Each gene = different dimmesnion
- After plotting – do dimmensional reduction to visualize all dimmesnions at once
Result - clustering of different cell tyes in a t-distrubuted stochastic neighbor embedding (tSNE) plot
- Compare how similar cels are to each other
- Cells = cluster based on similarity between cells
ScRNA seq in greater detail
Constrait of single cell RNA sequencing
- Need to know the sequence of the genome and the transcriptome to know what gene the transcript came from
- Can’t look at data for 1 cell need popultion data
Example single cell RNA sequencing experiment
Chart shows Expression level of 30 genes for each of 700 cells
Y axs = genes
X Axis = Different cells
Chart = see two types of cells that correspond to two types of genes
NOTE - Data for any 1 gene and any 1 cell is noisy because not doing deep reads BUT when agregating there is a clear pattern
Single Molecule Sequencing
Includes:
1. PacBio
2. Oxford nanopore
PacBio
Overall - Sequencing of a single DNA molecule by monitoring the binding of fluorescent nucleotides to a tether DNA polymerase
Process:
Have thousands of primed single molecule temples on slide + each polymerase is tether to the slide in a microwell –> when correct base is added by polymerase the correct base stays longer than the incorrect base –> correct base genrates a higher floruescent signal –> higher signal is considered the true signal
- Once incorporated flourophore on the terminal phosphate will be kicked out
Issue in PacBio
High (10%) error rate
- To get around issue – sequence many copeies of the same things –> can take average and know the true seqeucne
NanoPore sequecning
Overall - Sequencing of a single DNA molecule by threading the DNA through a transmembrane pore and monitoring the effect on Ionic conductance
Process - Thread DNA through a protein chanel in a membrane –> As teh DNA goes through the ions through through the pore give a signal
- Signal = based on a sequence of 5-6 bases sitting in the pore that keep ions from flowing
Pros and Cons of NanoPore
Issue:
1. High (10%) error rate
2. Need a lot of computing power to know what the patter of signal is
Pro:
1. Small machine
- Can be plugged into smart phone –> can do analysis in feild
- Example - Can sequence bacteria if someone has infection
MethylIC sequencing (mCseq) - Bisulfate sequnecing
Overall - Finds methylated C in DNA
- Methylated C = importnat epigenetic mark
Cytosone treated with bisulfate –> bisulfate reactes with the carbon to promote deaminatpion of the amino group –> forms Uracil
- Methulated C won’t recat ith bisulfate = no conversion to U
Process - Treat DNA samlpe with bisulfate –> Sequnce
Bisulfate Sequencing Results
Anywgere where a C was in the sequnce was deminataed to a U –> read as a T in PCR (Get T readout paired with A)
Compare sequence from the treated sample to an untreate sample sequence:
- C in the untreated and T in the treated = means the C was unmethylated
- C in the untreated and in the treated = was methylated
ATAC-seq
Overall – Finds accessible chromatin (areas in the genome more prone to opening) using a bacteria transposase that has preloaded DNA
Process:
Use TN5 tranposase –> tranpsosase sees that there is an opening in the DNA –> Tranposase does tranposition reaction –> tranposes the green and the red at thare at the end of the tranposon
- Tranposes has green and red oligios preloaded (See in image)
- Tranposase can’t hit DNA on nucleosome
- Tranposase can still insert transposon even if there are TF bound to open DNA because TF won’t take up all of the DNA
END – DNA insterted has the red and gree inserted on the ends –> can PCR amplify with primers for the red and green sequences –> see the regions of the genome that were accesble to the tranposase reaction
mRNA + ATAC-seq + bisulfate results
Chart - Genome brower caparisons of methyl CpG vs. Accesible chromatin (ATC-seq) and RNA abundence among the brain + liver + lung + kideney
- Bottom right blue line = shows exon structure of gene
- Each horizontal line = alignment of sequence to genome
- Verticle lines = histogram of the number of reads to each place
RNA data:
- Blood vessels in brain = has many reads of RNA (RNA transcripts) at the end of brain
ATAC-seq data: Shows regions of enhnacers that control gene expression by looking at reads with transpoons inserted
- Blue peaks in ATAC seq = regions of open chromatin
Methyl Seq data - shows regions where methylation is promienent
- Size of bar = percent of C that is methylated
- Peaks in ATAC-seq (regions with open chromatin) = areas with methylaion in lung adn liver BUT in the brain there is less methylation = characteristic of enhnacers
Chromosome confirmation cpature (3C, 4C etc.)
Overall - Long genome is condesed into 2 microns in non-random manner
- Have region that are far linearly on chrosmome that are close spatiallly
Process: Cross link DNA –> Cut DNA with Restriction enzymes to get fragemnts –> Ligate the ends of DNA back together –> Reverse crosslink –> Process ligated psices (varies depending on method) –> Product quanitofication
- Sequencing shows places where two seqeunces in different regions are linked togetehr more frequently than expected by chance = means the regions exist close together in the nucleus
- Can see that Blue and red sequences are close in 3D space because they ligated together = they interact in genoe
Issue – Need to know genome
Chromatin Immuno-precipitation and seqeunce (CHIP-Seq)
Use - Identify proteins bound to DNA/the sequence the proteins are bound to
Process – Cross link proteins to DNA –> fragment chromasome peices –> use AB to bind to the TF –> Purify the protein/DNA complex –> reverse corss linkes to seperate the DNA and protein –> Amplify and Sequence that were enriched by the captire
Example use - Target Histone markers using AB for histone marks –> Find chromatin with H3K4 –> find places enriched for mark in genome
CHIPseq chart results
Chart - Individual sequencing reads aligned to the genome following CHIP-seq with AB for histone modification or DNAse I seq
DNA as a barcode Process
Identify bacteria by looking at seqence of rRNA
Chart – 16S rRNA on X-Axis ; Y- Axis shows variability
- See varaibility in 16S rRNA across organism (peaks are variable) BUT also see regions withour any peaks (red boxes) that are coserved in ALL bacteria
Process - Use the conserved regions as places to anneal primers (facing towards each other) –> amplify the DNA within the procers –> sequence the middle varaible region
END – match the sequnce of sample to a known organism
DNA as a barcode to monitor enhancer expression
Function - DNA barcodes to monitor the expression level of each member of a library of cis regulatory elements
- want to know which enhnacer is active
- IMAGE - CRE = enhancer
Process:
1. Make barcoded oligionucelotide library + Minimal promotter and reprter gene –> put both into plasmid to make library hooked up to reporter (have DNA next to the reporter that distiguishes each of the different Cre)
2. Put plasmid into cell
3. Allow gene exprression to occur depending on if Cre works or not
- Active genes makes a lot of mRNA from reporter and its barcode vs. No active Cre then no mRNA
4. Seqeunce RNA –> catalogue abdundence of barcodes = can know the activity of cis-regulatory elements
Northern and Southern blot process
Process - Have sample –> Extrac RNA or DNA –> Run sample of DNA or RNA on a gel –> Blot gel to nitrocellulose memebrane (transfeer RNA or DNA to membrane) –> immobilize DNA o the nitrocellulose memebrane –> reveal the sequence of using a flourescnet ot radioactive probe –> Visualize labels RNA/DNA
Molecular Beacons process
- Start – Have molecular beacon
- Molecular beacon = peice of DNA where part of the seqeunce can form BP with the other end of the beacon (has ssDNA in loop next to pairs)
- Beacons can be in he dsDNA form with loop or in ssDNA form (linear)
- Beacon has a flouraphore at one end that can be flourescing when linear OR quenched when in loop - Extended DNA to maximum it can hybirdize to target (Hybridzing the loop forming region and non-loop forming region)
- Molecular beacon = peice of DNA where part of the seqeunce can form BP with the other end of the beacon (has ssDNA in loop next to pairs)
- Add probe to unknown sample of DNA and allow it to hybridize –> measure flourscence –> know if the target of loop region is present in unknown DNA
In Situ Hybirdization
Use - detect DNA or RNA in sample of tissue
Ways in situ can be done:
1. Microdissectiion
2. In situ hybridization
3. In situ sequences
4. RNA scope
In Situ Hybirdization (with florusence)
Overall – visualize flouresecnt probe with a microscope
Process – Prepare sample –> in situate hybridzation (done in cell) –> Flourescent microscopy
Example – Done in human chromosomes to visualize BCR-ABL chromosomal tranlocatiion
- See green and red signals next to each other (Shoud not be near eachother BUT here they are because have a chromosomal translocation)
- Used to diagnose leukemia
In Situ Hybirdization (with florusence) exmaple #2
Example 2 – In situte hybridization to tissue (dropsphilla embyro)
Image - See different color probes = can see striped patterns of gene expressio in embryo
RNA scope
Use - amplifies signals
Process:
1. Start - Use Z shaped probes
- Probes = have 10 BP of ssDNA at the top) ; 10 BP linker + 10 BP at the bottom
2. 10 BP at the bottom of the probe = hybridize to RNA ; 10 BP at the top bind to preamplifier probe
- Light blue and dark blue probes bind next to each other on the RNA = ONLY when you hybridize ALL 20 Bp is it long enough to form a stable hybrid with he preamplifier (DNA seqeunce)
- IF any proves bind non-specfically to the slide = not enough to give signals = the two Z probes need to be right next to each other and the only way to be right next to each other is if they bind to the mRNA = know amplifying the right signal
In Situ sequencing
Use - find mRNA sequence and mRNA location
Process - Collect cells in tissue context –> padlocks-based RCA –> sequencing by ligation
1. Have surface that are split into zone each with a specifc DNA sequnece that have oligiodT primer and a barcode
- glass slide with oligionucloetides sticking up
2. Put surface of mRNA slide on tissue –> mRNA in tissue binds to oligionucleotides on each size of the slide
- Barcode = tells you where the spot was
3. Take the mRNA capture and RT to cDNA
- cDNA wil contain the marcode sequences and the mRNA sequence
4. Sort by barcode –> see that at one barcode location there is X many copies of an mRNA 1 or X many copies of mRNA 2
Microijecting with needle
Overall - Inject cell with needle
Example – Microinjecting DNA into a fertilized egg using a large suction pippete to hold and egg and a thin needle to inject a pronucleus with cloned DNA
Process – Glass needle pnctures and injects the DNA
Cationic Lipids
Overall – Transfection of cells in culture with RNA or DNA that has been complexed in cationic lipids
Process - DNA + liposome forms lipoplex –> lipopoplex goes to lipid memebrane using endocytosis –> movement through memebrane forms endosome –> endosome matures to become a lysosome OR lipids mix in endosome and DNA escaose
- Cationic lipids coat the DNA because negatively charged DNA binds to the lipids
PCR assmebly
Purpose – Use PCR to assemble segments of DNA
- Example – If need RE sites in a certain place
Process:
1. Start - Have a series of overlapping fragments –> in a PCR they would anneal to each other
- Need 5 bases overlap + high fidelity polymerase
2. Get PCR extension of seed oligionucelotides
3. Insert primers unique to ends (green arrows)
4. PCR amplificatoon of target seqeunce
5. Get product where all of the segments are annealed o each other
Gibson Assembly
Purpose – Assemble DNA fragments together without relying on RE
- In vivo method
Process – have overlap between fragments –> add gibson assmebly master mix (reaction enzmes that contain repair enzymes) –> Fregments are annealed –> in vivo DNA pilymerase cloases the gape + ligase seals nicks
Short Hairpin RNA and RNA inhibition
Purpose – Decrease expression of mRNA
Process – have RNA template –> RNA is exported from the nucleus –> DICER proccesses the RNA further –> 1 strand of the dsRNA gets loaded to the RISC complex –> RISC-Ago + ssRNA fonds complementary RNA target –> RISC complex interacts with the mRNA product –> causes cleavage of mRNA –> decreases RNA expression
- Transfect RNA into cells = get knockdown
- Knockdown NOT KO
Use of RNAi
Example - Western blot showing short interfering RNA (siRNA) knockdown of a protein
- Shows western with different siRNAs against target
- Scrambled sequence siRNA = Negative control
- See siRNA1 and siRNA2 = RNA is there BUT rediced = knockdown
- NOT 0 expresion because trasfection is not 100% and cleavage is not 100%
NOTE - siRNA is a short double stranded RNA segment that becomes bound to the RISC complex (with release of one RNA strand)
- siRNA-1 and siRNA-2 are specific for the target mRNA
CRIPSR in Bacteria
CRIPSR is a form of genetic memory used to defend against a viral infection
Process - Viral sequence is cleaved and storred in memory cassete in the bacterial genome (Adpatation) –> viral genome is transcribed to make a gRNA that joins with cas9 protein (cRNA biogenesis) –> cas9 will target DNA seqeunces (targeting of mobile genetic eleemnts) –> Regulation of gene expression
- IF virus infects the cell again = DNA would be cleaved by the cas9/gRNA complex
Cas9 Protein
Cas9 protein – uses a gRNA to direct dsDNA breaks
- Often repairs dSDNA break with NHEJ –> NHEJ often has errors (often small deletion)
Image – cas9 enzyme (blue) is bound to gRNA (pruple) –> cas9 cleaves of two strands at the target
Using CRIPSR to add template DNA
Process - Add cas9 and gRNA in mice AND add PCR product of the 1 kb segment that overlaps with the cut
- Because added template = NOW teh cells will do HDR = get perfect rapir with the DNA that you wanted instered
- Template added has the mutations that you want inserted into the genome
Example use – Get mouse models with human mutation
Dead cas9 (dcas9)
Overal – muatate the cleavage active site of cas9 = mutant binds to sgRNA and traget DNA BUT can’t cleave DNA
Use:
1. Can Add activator domain to cas protein –> get activation of a gene
2. Can add repressor – get gene repression
3. Can add floruscent protein = will be able to see where on the chromsome the complex is bound
Phage Display process
Process - Encode in a segment of DNA that codes for a protein within the phage genome –> DNA will be inseerted into the viral genome as part of viral protein that is expressed on the sirface of the virus = can seeprate the virsues that express the protein by binding them to a target –> Only the proteins that bind will remain (rest are washed away) –> elute off the protein –> infect E.coli –> Repeat cycle of enrichment
- Get the protein of interest by immobilizing the virus
- Sequnece the virus that binds well
Example use - To seperate a protein (often seperarate the high binding afffinity protein)
- Immobilize the protein on the surface AND use phage display t increase AB specificity
PhIp Process
Overall – Create library –> Screen serum –> Anylze data
Process - Virus has peptide library on the surface that includes all of the proteins expressed in the human genome ; AB from a person captures a subset of virus –> sequence = can get ctaalogue pf proteins seqeunce from human genome from which the AB bind
- Works with confirmation sensotove binds
Overall - Put on the plate with all the antibodies, see which bind –> Sequence, see which peptides the person is reacting too
Mass Spectometry
Overall - Measure the mass to charge ratio of unknown molecles
Use - Protein identification
- Often done after digestion with protease (ex. Trypsin)
Process:
1. Have mix of proteins whose idetity you want to know –> fragment proteins with Trypsin –> ion fragment –> spend out at hight speed through magmnet
2. Charge partficles moving through the magnetic feild will bend
- BEND is at a different angle depending on the mass/charge ratio = Movment of particle chnages if have different mass to charge ratio
3. END – Can measure the mass to chart ratio to know how much material was there
Tandem Mass Spectometry Process
Process – Take sample –> Ionatzation with ESI/MALDI –> m/z seperation –> get MS1 precursor ion –> fragmentation (with CID) –> m/z seperation –> get MS2 product –> delection
- After 1st seperation = get peakd of 1 m/z ration may have multiple pepetdes = want to take the peaks and refragment it
- Refrgraent using chemical fragemnts –> can THEN seperate the fragments again
END – reasemble fragments based on the overhanging nature if the fragments
Proximity labeling
Use - Find proteins nearby protein of interest
Process - Fuse your protein with promiscuous derivative of BirA catalytic domain –> covalently links biotin to nearby proteins (non-specific) –> Capture biotinylated proteins using streptavidin beads –> Identify captured proteins with mass spec
