Purines
Adenine, guanine
Pyrimidines
Cytosine, Thymine, Uracil
Chargoff’s rules
- base composition of DNA varies from species to species
- DNA samples from different tissues of the same species have the same base composition
- DNA base composition does not change with age, nutritional state, environment
- (A=T, G=C)
H-bonding in DNA relative strengths
G-C bond stronger b/c 3 H-bonds (A-T has 2)
DNA Major grove
-Where nucleotide sequence is primarily read by DNA binding proteins
DNA length per turn
10.5 base pairs in a complete helix turn
When studying a virus, discovered that it is 19% adenosine, 32% thymidine. What does this tell you about the virus?
- is DNA, has thymidine
- adenosine is not equal to thymidine, so it cannon be double stranded DNA
- therefore is ssDNA
Functions of DNA
- long term storage for genetic info
- template for DNA replication
- coding for proteins and functional RNAs
DNA vs RNA stability
- DNA very stable, RNA/proteins are not (have to be environmentally sensitive)
- RNA unstable by design, hydroxide anions deprotonate 2’OH, which attacks phosphodiester bonds
- 2’ and 3’ cyclic monophosphates converted to 2’ and 3’ monophosphates
RNA 2˚ structure
- single stand, folded back on self
- hydrophobic bases stacked within helix
- fully/partially base paired
- antiparallel
- helices, internal loops/bulge (dsRNA separates due to lack of watson-crick base pairing), hairpin loop (most common, unpaired bases at loop)
- actions of functional RNA governed by the 2˚ structure
- can have non-watson-crick pairings (ie. G-T)
- can have base-triple interations (3-BP interaction)
What chemical interaction is NOT involved in stabilizing the transition of ssDNA into a double helical structure?
- Phosphodiester bond
- involved in the structure of DNA (backbone), but the helical structure is NOT stabilized by covalent interactions
Interactions that result in DNA double helix stabilization
H-Bonding
-A-T has 2 H-bonds, G-C has 3
-areas where helicase has to act usually has A-T rich regions due to weaker interaction between A and T
Hydrophobic+Van der Waals
-bases are hydrophobic, align to inside of mlc
Ionic interactions
-backbone highly neg. charged at neutral pH, electrostatic repulsion
-addition of salt neutralizes -ve charge
What would be the consequence of adding salt to a solution of dsDNA?
It would take a higher temp to melt. The helix will be stabilized by the cations
To promote DNA melting (denaturation)
- increase temp
- reduce salt
- add organic solvents (ie. urea)
- add DNA binding proteins (ie. helicase)
Quantification of nucleic acids
- nitrogenous bases absorb light (conjugated)
- use beer’s law (A=ecl)
- order of UV absorption: free dNTPs>ssDNA>dsDNA
Melting point (Tm)
-temp where half the DNA is denatured
Internal factors the influence Tm
-Base composition (G-C stronger than A-T)
Tm=0.41x(%G+C)+69.3˚C
-true for long DNA segments
-Salt concentration of 0.2M
External Factors that influence Tm
- High pH (causes deprotonation of bases, reduced H-bonding)
- addition of organic solvents decreases Tm
- addition of salt increases Tm
DNA hybridization
- separated DNA strands can renature with other polynucleotides sharing complementary sequences
- ie. cross species, DNA-RNA, DNA-DNA
- the stability of the hybrid depends on the stringency of the conditions
Stringency
- The strictness with which Watson-Crick pairing is required for nucleic acid hybridization under specific conditions of temp, pH, [Salt], etc.
- high stringency: hybridization occurs only when the two strands are highly compatible
