L5 Techniques of Molecular Biology: Analysis of DNA-Protein Interactions

Question 1

how can you determine if/where a protein binds to DNA?

Answer 1

1. Electrophoretic mobility shift assay (EMSA) - in vitro assay
2. chromatin immunoprecipitation (ChIP) - in vivo assay

Question 2

EMSA - how does it work?

Answer 2

• mix radioactive labeled probe DNA with a purified protein
• perform gel electrophoresis
• physical interaction slows the electrophoretic migration of DNA
• free DNA at the bottom

Question 3

ChiP - how does it work

Answer 3

• use formaldehyde
• isolate and shear DNA into small fragments
• add antibody and immunoprecipitate
• purify DNA
• test for enrichment with PCR

Question 4

ChIP - why use formaldehyde?

Answer 4

• when protein and DNA interact, they are not covalently bonded and thus dynamic
• to see interaction, need to chemically fix the protein to the DNA

Question 5

ChIP - what is formaldehyde?

Answer 5

• a cross-linking agent
• it forms true covalent bonds between DNA and protein
• does it in a way that transcription factors will not fall off

Question 6

ChIP - add antibody and immunoprecipitate

Answer 6

selectively pulls out specific transcription factor and its associated DNA

Question 7

ChIP - purify protein

Answer 7

• after getting targeted DNA
• wash everything else away (including transcription factor)

Question 8

ChIP - test for enrichment with PCR

Answer 8

• do PCR on DNA mix
• using primers with specific promoters of a gene A
• if the transcription factor does bind to that gene
• that DNA fragment is enriched within the DNA mix (can be confirmed with semi-quantitative or quantitative PCR)

Question 9

ChIP -Seq - what is it and how is it different from ChIP

Answer 9

• high throughput sequencing
• like ChIP but looking at many/all of the DNA that is enriched not just one

Question 10

ChIP -Seq - what do the results look like?

Answer 10

• need to compare it to a reference gnome
• can look to see more or less enrichment based on comparison