Lecture 26: Functional Genomics 2; Protein-Nucleic Acid Interactions

Question 1

What and why protein-nucleic acid interactions?

Answer 1

1. "These interactions are critical to 
    understand mol biol
2. Transcription, recombination, DNA 
    repair, spicing, etc ALL require a 
    protein to recognise particular DNA:
   a. structure
   b. sequence
3. Ensures correct temporal and spatial 
    occurrence of event

Question 2

What is EMSA?

Answer 2

1. In vitro technique
2. EMSA = “Electrophoretic Mobility-Shift
   Assay”
3. If DNA has a protein bound to it,
   migration through gel is slower than
   unbound DNA

Question 3

What is the process of EMSA

Answer 3

1. Label double-stranded DNA 
   (oligonucleotide with proposed binding 
   element)
2. Incubate DNA probe with protein
3. run native PAGE or agarose gel

Question 4

What is the process of EMSA

Answer 4

1. Label double-stranded DNA 
   (oligonucleotide with proposed binding 
   element)
2. Incubate DNA probe with protein
3. run native PAGE or agarose gel

Question 5

What are the pros and cons of EMSA

Answer 5

+ Can be used to monitor multiple proteins
interacting with the same DNA
+ Different proteins binding can be
determined by the size shift

• Do not reveal which sequence in the DNA
  the protein binds to (although can make
  mutations in the DNA seq and check if
  binding still occurs

Question 6

What is DNA Footprinting?

Answer 6

1. In vitro technique
2. Also called “nuclease protection
   footprinting
3. Allows identification of the DNA site
   (motif) bound by protein

Question 7

What is the DNA Footprinting procedure?

Answer 7

1. Label DNA
2. Bind protein to DNA
3. DNase (mild)
4. Remove protein
5. Denture DNA
6. After digestion = ELECTROPHORESIS

DNA bound by protein wont be ‘banded’ causing a gap in the ladder

Question 8

What are the pros and cons of DNA Footprinting?

Answer 8

+ Immediately identifies DNA binding site

• The protein needs to have a strong
  affinity for DNA

Question 9

What is chromatin immunoprecipitation (ChIP)?

Answer 9

1. In vivo technique
2. Powerful tool to determine protein-DNA
   interactions in a living cell
3. DNA resulting from ChIP can be identified using:
   a. PCR
   b. Microarrays (ChIP-on-chip)
   c. Sequencing (ChIP-seq)
4. ChIP-on-chip and ChIP-seq can tell you
   where in the entire genome a protein
   binds (lots of info)

Question 10

What is the process of chromatin immunoprecipitation (ChIP)?

Answer 10

1. Crosslinking of protein to DNA (formaldehyde or UV light)
2. Fragment chromatin (sonication)
3. Immunoprecipitation using antibody
4. Reverse crosslinking. Purify DNA

Question 11

What is the procedure fir ChIP-on-chip?

Answer 11

1. cells exposed to a cross-linking agent
2. cell lysis and sonication procedures;
   fragmented, soluble chromatin
3. immunoprecipitation (Ab’s)
4. Reverse crosslinks
5. Label with fluorescent dyes
6. Hybridisation on microarray

Question 12

What is the procedure for ChIP-seq?

Answer 12

1. Fragment chromatin immunoprecipitation
   2a. Non-histone ChIP
   2b. Histone ChIP
2. Reverse crosslinking. Purify DNA
3. Next gen (deep) seq

Question 13

What are the pros and cons of chromatin immunoprecipitation (ChIP)?

Answer 13

+ V. powerful
+ in vivo binding

• Similar to EMSA; limited resolution with
  proteins which bind to DNA weakly
• Expensive
• Need an Ab for your protein of interest