01. Molecular Structures — Nucleic Acid Structure and Hybridization

Question 1

What is hybridization?

Answer 1

When two complementary strands of single-stranded nucleic acid bond together to form double-stranded DNA/

Question 2

What factors denature DNA?

Answer 2

• Heat — sufficient heat will break most chemical bonds
• Low salt concentration — negative phosphate backbones will repel each other
• High pH (basic conditions) — bases will strip protons between electronegative centers
• H-bond influences
  
  • competition from molecules that can also form H-bonds
  • covalent modifications that modify electronegative centers
• Solubility increases
  
  • anything that makes hydrophobic substances more soluble.

Question 3

How do we monitor DNA denaturation?

Answer 3

• Core principle: dsDNA and ssDNA have different properties. We’re most interested in absorbance:
• ssDNA absorbance: high (hyperchromic)
  
  • bases are unstacked
• dsDNA absorbance: low (hypochromic)
  
  • bases are stacked

Question 4

What is melting temperature and what influences it?

Answer 4

• T_m: melting temperature; the temperature at which 50% of DNA is denatured.
  
  • Higher GC content = higher T_m because 3 H-bonds instead of 2.
  • Higher salt = higher T_m

Question 5

What factors affect renaturation?

Answer 5

• DNA concentration — more strands make it easier for complements to find each other
• Salt concentration — greater ionic conditions will mask repulsive forces between phosphate backbones
• Temperature — recombination occurs 20° — 25° C below T_m
• Size — longer strands take longer to renature
• Complexity — simple sequences renature faster

Question 6

What is complexity?

Answer 6

• The number of nucleotides in a non-repeating sequence + nucleotides in ONE COPY of a repeating sequence.
• The better definition is: the size of functional and non-repetitive section of a genome

Question 7

What is a C_°t analysis?

Answer 7

• Uses the rate of renaturation to measure DNA/genome complexity
• If two DNA sequences do not have repetitive elements + have similar GC content, their size is proportional to their C_°t_½
• Features
  
  • A Cot plot can have 3 bumps
    
    1. Fast — short, highly repetitive sequences that renature quickly
    2. Medium — less repetitive, longer to renature
    3. Slow — unique sequences, longest to recombine
  • For human:
    
    • Fast — unknown roles
    • Medium — gene families, non-coding functions
    • Unique — protein coding

Question 8

Determine genome size with a Cot plot.