Methods

Question 1

Checking for DNA sequence homology

Answer 1

Past: low-stringency DNA probes
Now: BLAST analysis

Question 2

Checking for structural homology

Answer 2

Immunoprecipitation with antibodies

Question 3

Checking fo functional similarity

Answer 3

wt w/ non-coding mutations plasmid complementation with KO, siRNA knockdown (if essential) or thermosensitive mutant.

Control: multiple siRNAs

Question 4

Checking for DNA damage proteins

Answer 4

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.

Question 5

Detecting Protein-protein interactions

Answer 5

1. Co-IP: Cell extract incubated w/ antibody. Control: Cell extract w/out antibody. Cell extract w/ different antibody.
2. Y2H
3. BioID screen: using promiscuous biotin ligase.

Question 6

Determining cell cycle phase

Answer 6

FUCCI w/ FACS. RFP-tagged cdt1, GFP-tagged Geminin. cdt1 needed pre-S, Geminin needed post-G1.

Question 7

Determining telomerase function

Answer 7

Have telomere imitating oligonucleotide. Elongated by telomerase, then PCR, and run on Southern blot to determine extent of elongation.

Question 8

Western Blot

Answer 8

Protein detection. Use ab

Question 9

Southern Blot

Answer 9

DNA detection. probe

Question 10

Northern blot

Answer 10

RNA detection

Question 11

Eastern Blot

Answer 11

detection of Post-translational modification. Multiple Abs.

Question 12

Interference screen

Answer 12

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)

Question 13

Synthetic lethality screens

Answer 13

Apply 2D siRNA library. Control: no siRNA, 1D siRNA screen

Question 14

Check that a tag has not affected enzyme function

Answer 14

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.

Question 15

In vitro DNA binding assay

Answer 15

Radiolabeled DNA fragment -> electrophoretic gel

Question 16

In vivo DNA binding assay

Answer 16

Chip-seq.

Question 17

Chip -seq

Answer 17

1. crosslink
2. sonicate
3. Immunoprecipitate
4. Mass spec. proteins & sequence DNA

Question 18

Detecting native protein size

Answer 18

• size exclusion chromatography: smaller molecules enter bead pores, take longer to elute.
• sucrose gradient ultracentrifugation.

Question 19

CRISPR screen steps

Answer 19

1. 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
2. Genetically modify cells to express Cas9 (Use YFP reporter to sort in FACS)
3. Transduce cells with sgRNA library and check for the phenotype
4. Extract the DNA for the cell with phenotype and sequence

Question 20

Checking for chromosomal changes

Answer 20

multicolored FISH: Can visualize amplifications (more color), deletions (less color), or translocations (change in distances between colors)

Question 21

How to measure DNA methylation?

Answer 21

Bisulphite modification:

C-> U – (taq polymerase) -> T
Cmet not affected
Compare sequences