Checking for DNA sequence homology
Past: low-stringency DNA probes
Now: BLAST analysis
Checking for structural homology
Immunoprecipitation with antibodies
Checking fo functional similarity
wt w/ non-coding mutations plasmid complementation with KO, siRNA knockdown (if essential) or thermosensitive mutant.
Control: multiple siRNAs
Checking for DNA damage proteins
Immobilized repair template Assay.
- Hybridize damaged dna w/ biotinylated probe. Capture pair with spreptavidin.
- Mix with cell lysate, and wash.
- ID proteins that remain attached to DNA with mass spec.
Detecting Protein-protein interactions
- Co-IP: Cell extract incubated w/ antibody. Control: Cell extract w/out antibody. Cell extract w/ different antibody.
- Y2H
- BioID screen: using promiscuous biotin ligase.
Determining cell cycle phase
FUCCI w/ FACS. RFP-tagged cdt1, GFP-tagged Geminin. cdt1 needed pre-S, Geminin needed post-G1.
Determining telomerase function
Have telomere imitating oligonucleotide. Elongated by telomerase, then PCR, and run on Southern blot to determine extent of elongation.
Western Blot
Protein detection. Use ab
Southern Blot
DNA detection. probe
Northern blot
RNA detection
Eastern Blot
detection of Post-translational modification. Multiple Abs.
Interference screen
Administer drug in ht-screen in combination with siRNA library. Look for interfering RNAs which increase drug effect.
Controls: No siRNAs, no drug (to avoid essential gene KDs)
Synthetic lethality screens
Apply 2D siRNA library. Control: no siRNA, 1D siRNA screen
Check that a tag has not affected enzyme function
Run enzymatic assay. Controls are enzyme without tag, and no enzyme. Demonstrate that there is there is extra phosphate, for example, via western or eastern blot.
In vitro DNA binding assay
Radiolabeled DNA fragment -> electrophoretic gel
In vivo DNA binding assay
Chip-seq.
Chip -seq
- crosslink
- sonicate
- Immunoprecipitate
- Mass spec. proteins & sequence DNA
Detecting native protein size
- size exclusion chromatography: smaller molecules enter bead pores, take longer to elute.
- sucrose gradient ultracentrifugation.
CRISPR screen steps
- Generate a library of sgRNA targeting genes of interest, each gene targeted by multiple sgRNAs to increase reproducibility
- Library can be designed to target few genes or whole genome, possible to study nc region - Genetically modify cells to express Cas9 (Use YFP reporter to sort in FACS)
- Transduce cells with sgRNA library and check for the phenotype
- Extract the DNA for the cell with phenotype and sequence
Checking for chromosomal changes
multicolored FISH: Can visualize amplifications (more color), deletions (less color), or translocations (change in distances between colors)
How to measure DNA methylation?
Bisulphite modification:
C-> U – (taq polymerase) -> T
Cmet not affected
Compare sequences
