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Human Genetics
DNA Makes RNA Makes Protein
Terminology Review
 Chromosome
 Threadlike structures in the nucleus that carry genetic
information
 Gene
 Fundamental unit of heredity
 Inherited determinant of aphenotype
 Locus
 Position occupied by a gene on a chromosome
 Gene
 sequence of DNA that instructs a cell to produce a particular
protein
 DNA
 Deoxyribonucleic Acid-the molecule that forms genes
 The genetic material
 Allele
 Different DNA sequences possible for the same gene location
“A genetic material must carry out two jobs:
duplicate itself and control the development of
the rest of the cell in a specific way.”
-Francis Crick
DNA (deoxyribonucleic acid) is
a chain of nucleotides
 Sugar: Deoxyribose
 Phosphate
 Base - one of four types:
adenine (A), thymine (T)
guanine (G), cytosine (C)
Which of these are Purine bases?
Pyrimidine bases?
 A (adenine)
C (cytosine)
T (thymidine)
G (guanine)
 A guy walks into a bar and says "My name's
Chargaff, and 22% of my DNA is "A"
nucleotides. I'll bet anyone that they can't
guess what percentage of my DNA is "C"
nucleotides!" You say "I'm thirsty, so I'll take
that bet!" http://escience.ws/b572/L1/L1.htm
DNA Bases Pair through Hydrogen
Bonds
Erwin Chargaff observed:
# of adenine = # of thymine
# of guanine = # of cytosine
Complementary bases pair:
A and T pair
C and G pair
Cytosine deamination (i.e. water attacks!)
DEAMINA
TION
--------->
Cytosine
Uracil
What's the difference between
DNA and RNA?
DNA contains the sugar deoxyribose
while RNA is made with the sugar ribose.
It's just a matter of a single 2' hydroxyl,
which deoxyribose doesn't have, and
ribose does have.
You all remember that RNA uses the
base uracil instead of thymine too.
Cytosine naturally has a high rate of
deamination to give uracil
If C-U deamination occurs and
then is replicated, the U will pair
with an A not the C with a G
DEAMINATION
--------->
Cytosine
Uracil
If 5-methyl C-T deamination occurs
and then is replicated, the T will
pair with an A not the C with a G
DEAMINATION
--------->
5 methyl Cytosine
Thymine
DNA is a Double Helix
C
G
T
A
G
C
T
A
C
G
A
A
T
G
G
C
T
A
C
T
G
C
 X-ray diffraction
indicated DNA has a
repeating structure.
- Maurice Wilkins and
Rosalind Franklin
 DNA is double-stranded
molecules wound in a
double helix.
 -James Watson and
Francis Crick
DNA Double Helix
T
C
G
P
A
P
5’ P
PP
P
P
C
A
T
C
P
P
G
P
G
P
P
C
G
P
3’
3’  A sugar and phosphate
“backbone” connects
nucleotides in a chain.
 DNA has directionality.
 Two nucleotide chains
together wind into a helix
 Hydrogen bonds
between paired bases
hold the two DNA
strands together.
 DNA strands are
antiparallel
5’
Orientation of DNA
The carbon atoms on the sugar ring are numbered for
reference. The 5’ and 3’ hydroxyl groups (highlighted on
the left) are used to attach phosphate groups.
 The directionality of a DNA strand is due to the orientation of
Structure of DNA
 Two nucleic acid chains running in opposite directions
 The two nucleic acid chains are coiled around a central
axis to form a double helix
 For each chain – the backbone comes from linking the
pentose sugar bases between nucleotides via
phosphodiester bonds connecting via 3’ to 5’
 The bases face inward and pair in a highly specific
fashion with bases in the other chain
 A only with T, G only with C
 Because of this pairing – each strand is complementary
to the other
5’ ACGTC 3’
3’ TGCAG 5’
 Thus DNA is double stranded
Chromatin = DNA and associated proteins
DNA winds around
histone proteins
(nucleosomes).
Other proteins wind
DNA into more tightly
packed form, the
chromosome.
Unwinding portions of
the chromosome is
important for mitosis,
replication and making
RNA.
Genes: molecular definition
A gene is a segment of DNA
 which directs the formation of RNA
 which in turn directs formation of a protein
The protein (or functional RNA) creates
the phenotype
Information is conveyed by the sequence
of the nucleotides
Why is DNA good Genetic
Material?
A linear sequence of bases has a high
storage capacity
a molecule of n bases has 4n
combinations
just 10 nucleotides long -- 410 or
1,048,576 combinations
Humans – 3.2 x 109 nucleotides long – 3
billion base pairs
Required properties of a genetic
material
Chromosomal localization
Control protein synthesis
Replication
DNA Replication
- the process of making new copies of the DNA molecules
Potential mechanisms:
organization of DNA strands
Conservative
old/old + new/new
Semiconservative
old/new + new/old
Dispersive
mixed old and new on
each strand
Meselson and Stahl’s replication
experiment
Conclusion: Replication is semiconservative.
Replication as a process
Double-stranded DNA unwinds.
The junction of the unwound
molecules is a replication fork.
A new strand is formed by pairing
complementary bases with the
old strand.
Two molecules are made.
Each has one new and one old
DNA strand.
Fig 8.14
Replication in vivo is complex
Replication requires the coordinated
regulation of many enzymes and
processes




unwind the DNA
synthesize a new nucleic acid polymer
proof read
repair mistakes
Enzymes in DNA replication
Helicase unwinds
parental double helix
DNA polymerase
binds nucleotides
to form new strands
Binding proteins
stabilize separate
strands
Exonuclease removes
RNA primer and inserts
the correct bases
Primase adds
short primer
to template strand
Ligase joins Okazaki
fragments and seals
other nicks in sugarphosphate backbone
Replication
3’
3’
5’
5’
3’
5’
Helicase protein binds to DNA sequences called
origins and unwinds DNA strands.
Binding proteins prevent single strands from rewinding.
Primase protein makes a short segment of RNA
complementary to the DNA, a primer.
3’
5’
Replication
Overall direction
of replication
3’
3’
5’
5’
3’
5’
3’
5’
DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.
DNA polymerases require an underlying template (and a primer)
and cannot synthesize in the direction 3' to 5'. That is, they
cannot add nucleotides to a free 5' end.
Replication
Overall direction
of replication
3’
5’
3’
5’
3’
5’
3’
5’
DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.
DNA polymerase proofreads bases added and
replaces incorrect nucleotides.
Replication
Overall direction
of replication
3’
3’
5’
5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
3’
5’
Replication
Overall direction
of replication
3’
3’
5’
5’
Okazaki fragment
3’
5’
3’ 5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
3’
5’
Replication
Overall direction
of replication
3’
3’
5’
5’
Okazaki fragment
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
3’ 5’
3’
5’
Replication
3’
5’
3’
5’
3’
5’
3’ 5’
3’5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication
3’
5’
3’
5’
3’
5’
3’5’
3’5’
3’
5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication
3’
5’
3’
5’
3’
5’
3’5’
3’5’
3’
5’
Exonuclease enzymes remove RNA primers.
Replication
3’
3’
5’
3’
5’
3’5’
3’
5’
Exonuclease enzymes remove RNA primers.
Ligase forms bonds between sugar-phosphate
backbone.
Replication
5’
3’
3’
5’
5’
3’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
5’
3’
5’
3’
3’
5’
5’
3’
5’
3’
5’
3’
5’
3’
5’
5’
5’
3’
5’
5’
3’
5’
3’
3’
3’
5’
3’
5’
3’ 5’
3’
3’
3’
5’
3’
5’
3’
3’
5’
5’
3’
3’
5’
3’
5’
3’
5’
General rules of conduct for DNA
polymerase I enzymes (like Taq)
1. Remember your base pairing rules: G
goes with C and A goes with T.
2. The 5' ends are strictly off limits
3. There will be no synthesis without a free
3' end
4. There will be no degradation without a
free 3' end
General rules of conduct for DNA
polymerase I enzymes (like Taq)
5. There will be no synthesis without an
underlying template
6. Under no circumstances may you make
a synthetic addition to the 5‘ end
7. There is no reconstruction of a broken
phosphodiester bond, unless you have
ligase. If you are synthesizing DNA and
run into an obstruction on your template,
you must stop and leave the nick
unrepaired.
General rules of conduct for DNA
polymerase I enzymes (like Taq)
8.If you have been provided with a free 3'
end, a template, and a substrate
molecule that is correct, you must add
that nucleotide to the growing end of the
strand (i.e. to the 3' end.)
PCR: Polymerase Chain Reaction
 Selective replication and amplification of
specific(targeted) DNA sequences.
 PCR basics
 Know some sequence of the piece of
genomic or other DNA to be amplified
 DNA primers - short DNA pieces of
sequences complementary to the DNA
sequence to be amplified
 Four nucleotide building blocks
 Taq1 - DNA polymerase, Buffer, MgCl2
Polymerase Chain Reaction (PCR)
Denaturation
DNA template is melted with
high heat to separate strands.
Annealing
Each DNA primer anneals, binding
to its complementary sequence
on the template DNA
Extension
DNA polymerase creates a
new strand of DNA complementary
to the template DNA starting from
the primer’s free 3’ end.
Multiple rounds of denaturation-annealing-extension are
performed to create many copies of the template DNA
between the two primer sequences.
Polymerase Chain Reaction (PCR)
DNA template is denatured with heat to separate strands.
5’
3’
G
C
5’
A
T
A
T
C
G
T
A
A
T
G
C
C
G
G
C
3’
Polymerase Chain Reaction (PCR)
DNA template is melted with heat to separate strands.
5’
3’
5’
G
A
A
C
T
A
G
C
G
C
T
T
G
A
T
C
G
C
3’
Polymerase Chain Reaction (PCR)
DNA polymerase creates a new strand of DNA complementary to the template
DNA starting from the primer.
5’
3’
G
A
A
C
T
A
G
C
G
C
T
T
G
A
T
C
G
C
5’
3’
3’
5’
5’
G
A
A
C
T
A
G
C
G
C
T
T
G
A
T
C
G
C
3’
Polymerase Chain Reaction (PCR)
Template Base Pairing
Requires correct temperature.
Too hot and nothing can form hydrogen
bonds.
Too cold and the template reforms and
the primers can form weak hydrogen
bonds with sequences that are not
perfectly complementary.
http://escience.ws/b572/L3/L3.htm
Genome and Epigenome vary in
Monozygotic Twins
 Identical Twins don’t actually
have completely identical
DNA
 http://www.cell.com/AJHG/abs
tract/S0002-9297(08)00102-X
 Bruder et al. Phenotypically
Concordant and Discordant
Monozygotic Twins Display
Different DNA Copy-NumberVariation Profiles AJHG, Vol
82, No, 3, 763-771
http://www.nytimes.com/2008/03/11/health/11real.html?scp=3&sq=epigenetics
&st=cse