The Structure of Nucleic Acids

Determining the primary structures of nucleic acids (the nucleotide

sequence) is now easier compared to sequencing of proteins due to
two factors:
a) discovery of restriction endonucleases
b) the power polyacrylamide gel electrophoresis

Two basic protocols for sequencing nucleic acids:

1. Chain termination or dideoxy method by F. Sanger
2. Base-specific chemical cleavage method by A.M. Maxam and
W. Gilbert
DNA replication yields two daughter DNA
duplexes identical to the parent DNA molecule.
Each strand in the parent DNA molecule is copied
in a complementary fashion to form a new second
strand by DNA polymerase.
Base-specific chemical cleavage method: cleavage at purines using
dimethylsulfate, followed by scission with piperidine
Base-specific chemical cleavage method: cleavage at pyrimidines
by hydrazine
To interpret the Maxam-Gilbert
sequencing of DNA:
1. there is a specific reaction for
G only
2. there is a purine-specific
reaction that removes and G so
that #2 - #1 = A
3. there is a reaction specific for
the pyrimidines (C+T)
4. if reaction # 3 above is run in
1-2M NaCl, only works only with C.
5. #3 - # 4 = T
Automated DNA Sequencing- can identify 104 bases a day
Human genome = 2.9 billion bp
Secondary structures of the DNA = A, B and Z

Stacking of the bases

Repetition of the helix

Major conformation of
DNA solution = B
DNA molecule is stable:
1. H bonding of the purines to the pyrimidines, H-bonding of the polar molecules in sugar-
phosphate backbone with water
2. Negatively charged phosphate groups are on the exterior of the helix so minimal effect on
each other and free to bind with Mg2+
3. The core of the helix which are the nitrogenous bases, aside from being H-bonded, are
also having hydrophobic interactions and van der Waals forces
The three
secondary
structures of the
DNA molecule
Intercalating agents distort the DNA ethidium bromide, acridine orange,
actinomycin D (aromatic flat, hydrophobic molecules with fused heterocyclic rings)
Denaturation of DNA = disruption of the hydrogen bonds by pH,
temperature (melting of the DNA, Tm), or ionic strength

Higher G+C content = higher

Tm bec. 3 H-bonds
Higher ionic strength =
higher Tm

Heat denaturation of DNA.

Tertiary structures of DNA = supercoils and cruciforms

Coiling in a spiral fashion around a

toroid

intertwining

Supercoils in long linear

DNA arranged into loops
and ends are restrained
(chromosonal DNA)
Cruciform another tertiary structure
-happens if sequence of DNA is palindromic