Introduction to DNA Cloning
Nucleotides and DNA Structure
Learning Objectives
• Understand the double helix structure
and dimensions of DNA molecule.
• Understand the chemical bonds of the
DNA molecule (which are covalent and
which are hydrogen).
• Understand complementary base-pairing
rules of the DNA molecule and be able
to predict the opposite strand.
• Understand the antiparallel nature of
DNA molecule.
DNA History
• Deoxyribonucleic acid, or DNA, was
discovered in the late 1860s.
• It was ignored because it seemed too simple:
A,C, G, T.
– That’s because they degraded it when they
purified it.
• In the 1940s scientists discovered that
chromosomes, which carry hereditary
information, consist of DNA and proteins.
• Experiments conducted throughout the 1940s
showed that DNA actually seemed to be the
genetic material.
Happy 51th Birthday
DNA Structure
The first X-ray photograph of crystalline DNA in the A form.
Taken by Rosalind Franklin, 1952
Evidence for a Double Helix
• Rosalind Franklin, working with Maurice
H.F. Wilkins, studied isolated fibers of
DNA by using the X-ray diffraction
technique, a procedure in which a beam
of parallel X rays is directed on a
regular, repeating array of atoms.
• Watson saw pictures when Wilkins
showed them at a talk, without
Franklin’s knowledge.
Franklin’s Data
• The diffraction patterns obtained by
directing X-rays along the length of
drawn-out fibers of DNA indicated that
the molecule is organized in a highly
ordered, helical structure.
• The data showed DNA was a helical
structure which had two distinctive
regularities of 0.34 nm and 3.4 nm along
the axis of the molecule.
• It looked like a double helix.
Watson and Crick
• Linus Pauling built a model of an alpha
helix for protein structure and won the
Nobel Prize.
• Watson and Crick were inspired that
Pauling used his imagination and
molecular models to deduce this protein
structure.
• They believed if an eminent scientist
such as Pauling could model a structure
with little experimentation, then they
might be able to do the same with DNA.
What Watson and Crick Knew
Main components of DNA:
• Phosphates
• Sugars
• Four nitrogenous bases
–
–
–
–
Adenine
Thymine
Cytosine
Guanine
• Using wire and pieces of metal, Crick and
Watson began building scale models of
DNA.
Chargaff’s Rules
• Chargaff had found that the amounts of
adenine and thymine were approximately
equal and the amounts of guanine and
cytosine were also approximately equal.
• This information gave Watson and Crick
the idea that the bases might be paired
in a specific way.
Base Composition Can Differ
but Chargaff’s Rules Still Hold.
Organism
%A
%T
%G
%C
A+G A+T
T+C G+C
E. coli
bacterium
26.0
23.9
24.9
25.2
1.04
1.00
S. cerevisiae 31.7
yeast
32.6
18.3
17.4
1.00
1.80
Z. mays
corn
25.6
25.3
24.5
24.6
1.00
1.04
D. melanogaster
30.7
29.4
19.6
20.2
1.01
1.51
30.2
30.3
19.9
19.6
1.01
1.53
fly
H. sapiens
human
Failure at First
• At first, Watson imagined that the
bases paired like with like, for example
adenine with adenine, and cytosine with
cytosine.
• The resulting model did not fit
Franklin’s X-ray data.
• Then Watson and Crick discovered that
thymine and guanine could adopt a
slightly different chemical shape, and
their models used the wrong version of
the bases.
Success
• Using the new forms, Watson
discovered that he could make two base
pairs, one consisting of adenine and
thymine, and the other consisting of
guanine and cytosine, that had exactly
the same size.
• They built the model and wrote the
paper.
• Watson and Crick discovered the
structure (or solved it) without direct
experimentation themselves.
• They read, thought and talked their way
to a Nobel Prize.
The Solution
1. The DNA molecule consists of two
polynucleotide chains wound around
each other in a right-handed double
helix.
2. Viewed on end, the two strands wind
around each other in a clockwise
(right-handed) fashion.
2. The diameter of the helix is 2 nm or 20 Å
2 nm
The Solution (con’t)
3. The two chains are antiparallel
(= show opposite polarity).
• The two strands are oriented in
opposite directions with one strand
oriented in the 5' to 3' way, while the
other strand is oriented 3' to 5'.
Three components of each nucleotide
Four different nucleotides in DNA
Chemists name carbon atoms within the ring structures of
organic molecules: C1, C2, C3 etc. When there are two rings
in a structure, they name one ring “prime. In nucleotides,
the base is one ring (C1, C2 C3 etc) and the sugar was named
prime. The 5’ carbon has the phosphate and the 3’ carbon
has an OH group on the carbon.
The Solution (con’t)
4. The sugar-phosphate backbones are on
the outsides of the double helix, while
the bases are oriented toward the
central axis.
• The bases of both chains are flat
structures oriented perpendicularly to
the long axis of the DNA; that is, the
bases are stacked like pennies on top of
one another (except for the "twist" of
the helix).
The Solution (con’t)
5. The bases of the opposite strands are
bonded together by relatively weak hydrogen
bonds.
• The only specific pairings are A with T (two
hydrogen bonds) and G with C (three
hydrogen bonds).
• The A-T and G-C base pairs are the only ones
that can fit the physical dimensions of the
helical model.
• The specific A-T and G-C pairs are called
complementary base pairs, so the nucleotide
sequence in one strand dictates the
nucleotide sequence of the other.
The Solution (con’t)
6.The base pairs are 0.34 nm apart in the
DNA helix.
• A complete (360 degrees) turn of the
helix takes 3.4 nm; therefore, there are
10 base pairs per turn.
• Each base pair, then, is twisted 36
degrees clockwise with respect to the
previous pair.
0.34 nm 2 bp distance
3.4 nm, 10 base pairs per turn
The Solution
7. Because of the way the bases bond with
each other, the two sugar-phosphate
backbones of the double helix are not
equally spaced along the helical axis.
This results in grooves of unequal size
between the backbones called the major
groove (the wider groove of the two) and
the minor groove (the narrower groove of
the two).
Both of these grooves are large enough to
allow protein molecules to make contact
with the bases.
DNA and RNA as Chemicals
• Chemical Bonds
–
–
–
–
–
Covalent (permanent)
Ionic (salt)
Hydrogen (sharing H, very weak)
Van der Waals Bonds
Hydrophobic Interactions
Charge in Molecules
• Water is perhaps the most obvious
example of a molecule with partial
charges. The symbols delta+ and deltaare used to indicate partial charges.
Covalent Bonds
• Covalent Bonds are the strongest
chemical bonds, and are formed by the
sharing of a pair of electrons.
• Once formed, covalent bonds rarely
break spontaneously.
– Covalent bonds don’t fall apart when heated
or dissolved in a solvent like water.
Ionic Bonds
• Ionic bonds are formed when there is a
complete transfer of electrons from
one atom to another, resulting in two
ions, one positively charged and the
other negatively charged.
Hydrogen Bonds
• Hydrogen bonds are
formed when a
hydrogen atom is
shared between two
molecules.
Double strands of DNA are held
together by hydrogen bonds.
• The DNA molecule is usually doublestranded, with the sugar-phosphate
backbone on the outside of the helix.
• In the interior are pairs of nucleotide
bases, holding the two strands together by
hydrogen bonds.
• Hydrogen bonding between the bases is
specific. The adenine base can pair only
with the thymine base, and the guanine
base can only pair with the cytosine base.
Van der Walls Interactions
• Van der Walls interactions
are very weak bonds formed
between nonpolar molecules
or non-polar parts of a
molecule. The weak bond is
created because a C-H bond
can have a transient dipole
and induce a transient dipole
in another C-H bond.
H
H
| ~~~~ |
CH3 CH3
Hydrophobic Interactions
• Nonpolar molecules cannot form Hbonds with H2O, and are therefore
insoluble in H2O.
• These molecules are known as
hydrophobic (water hating), as opposed
to water loving hydrophilic molecules
which can form H-bonds with H2O.
If thymine makes up 15 percent
of the bases in a certain DNA
sample, what percentage of bases
must be cytosine?
If thymine makes up 15 percent
of the bases in a certain DNA
sample, what percentage of bases
must be cytosine?
thymine = 15%, then adenine = 15%
A + T = 30%, then G + C = 70%
So, cytosine is 1/2 of 70% = 35%
A certain segment of DNA has the
following nucleotide sequence in one
strand:
5’ ATTGGCTCT 3’
What must be the sequence of the
other strand (label its 5’ and 3’ ends)?
A certain segment of DNA has the following
nucleotide sequence in one strand:
5’ ATTGGCTCT 3’
What must be the sequence of the other strand
(label its 5’ and 3’ ends)?
Writing in the same direction:
5’
Writing 5’ to 3’:
3’ TAACCGAGA
5’ AGAGCCAAT 3’
How many bases are there in 2 kb
(2000 base pairs) of DNA?
How many bases are there in 2 kb
(2000 base pairs) of DNA?
4000 bases in all.
For the DNA strand 5'-TACGATCATAT-3' the
correct complementary DNA strand is:
A 3'-TACGATCATAT-5'
B 3'-ATGCTAGTATA-5'
C 3'-AUGCUAGUAUA-5’
D 3'-GCATATACGCG-5’
E 3'-TATACTAGCAT-5'
Correct Answer is:
B 3'-ATGCTAGTATA-5'
5’-TACGATCATAT3’
3'-ATGCTAGTATA5’
This choice has the correct polarity and
complementarity.
DNA Structure as a Ladder
•The curving sides of the ladder of DNA represent
the sugar phosphate backbone.
•The rungs are the base pairs.
•The spacing between the base pair rungs is 3.4 Å
(Angstroms are 1-10 or 1/10,000,000,000 of a
meter or 1/10 nanometer).
•The helix repeat distance is 34 Å, meaning there
are 10 base pairs per turn of the helix.
•The strands are antiparallel. If one has 5’ to 3’
polarity from top to bottom, the other must have 3’
to 5’ polarity from top to bottom.
•The helix is 20 Å across at the base.