Download Essential Biochemistry

Answers in Studying Molecular Biology
How does human genetic information differ from
our closest relative?
FOXP2 that encodes
for a speech/language
development protein
split from the ape
linage 6 million years
ago
Answers in Studying Molecular Biology
Are humans genetically programmed to
contract certain diseases?
Healthy Lung
Cystic fibrosis with an
occurrence of 1 in 3,000 for
U.S. babies is caused by a
defective protein resulting in
high lung secretions
Cystic Fibrosis Lung
DNA: The Genetic Code
Deoxyribonucleic acid (DNA) was
identified in 1944 as the information
required to transform normal bacterial
strains to pathogenic ones
In 1951 DNA and not proteins was
confirmed as the infectious agent for
bacteriophages (viruses that infect
bacteria)
DNA: The Genetic Code
Chargaff’s rule (A+G = C+ T)
Watson and Crick DNA
structure elucidation (1953)
Bases that Constitute DNA
At what
position does
the purine or
pyrimidine
base link with
the ribose
sugar
backbone?
Nucleotides (NTPs) versus Deoxynucleotides (dNTPs)
RNA ribose sugar
DNA 2’-deoxyribose
sugar
What is the difference
between a nucleotide
and a nucleoside?
DNA Double Helix Stability
Base π stacking and
Base pair H-bonding
DNA Structural Features
Anti-parallel
polynucleotide strands
Right-handed helix
Sugar-phosphate
exterior exposed to
solvent
Mg+2 stabilizes
phosphate groups
Two unequal grooves
Axial View of DNA
Sugar-phosphate
exterior exposed
to solvent
DNA Melting Curve
UV absorption greater
with double- versus
single-stranded
DNA
DNA Re- and De-naturation
DNA can renature with
single strands
re-forming a
double helix if
cooling occurs
slowly.
In nature, DNA can be unwound by helicases and cut by
endo- or exo-nucleases at phosphodiester bonds
DNA Melting Point Depends on GC Content
RNA versus DNA Components
Sugar hydroxylation pattern
Base methylation pattern
RNA Structural Features
Less stable
Normally single stranded
Can fold and base pair with complementary
segments of the same strand
Transfer RNA molecule
Other Nucleotides Molecules/Functions
Acetyl unit transferase
+ R-C(O)CH3
Other Nucleotides Molecules/Functions
Oxidation/Reduction Reactions
Other Nucleotides Molecules/Functions
Oxidation/Reduction Reactions
Central Dogma of Molecular Biology
DNA, RNA and proteins, are linear polymers (i.e. each
monomer is connected to at most two other monomers)
The monomer sequence effectively encodes information
DNA Replication
Proteins catalyzed:
[i] unwinding of the super helix –
DNA helicases
[ii] unwinding of the double helix –
DNA helicases
[iii] replicate the master template –
DNA polymerases
DNA Transcription
RNA polymerase and transcription factors catalyze
messenger RNA (mRNA) formation from DNA sections
In eukaryotes the primary transcript (pre-mRNA) is
processed via alternative splicing
Nucleotides (NTPs) versus Deoxynucleotides (dNTPs)
RNA ribose sugar
DNA 2’-deoxyribose
sugar
What is the difference
between a nucleotide
and a nucleoside?
RNA to Protein Conversion: Translation
Mature mRNAs attach to ribosomes where it is read as a
triplet codon
Initiation and elongation factors bring aminoacylated
transfer RNAs (tRNAs) into the ribosome-mRNA complex
Standard Amino Acid Genetic Code
Cystic Fibrosis
Protein Function/
Gene Structure
Diagnostic signs of
CF include:
High chloride
concentration in
sweat
Thick mucus in the
airways
A Mutation Site Resulting in Cystic
Fibrosis
Sanger or Dideoxy DNA Sequencing
Components for the Chain Termination Method:
• DNA fragment for sequencing
• Primers
• dNTPs
• ddNTPs
Sanger or Dideoxy DNA Sequencing
What direction does the DNA
polymerase elongate the DNA
fragment?
Sanger or Dideoxy
DNA Sequencing
Migration smaller is faster
through the gel (bottom smallest)
Each peak represents 1 base
Pyrosequencing
Based on DNA synthesis
Template DNA
is immobilized
Peak area
represents
base number
http://www.pyrosequencing.com/DynPage.aspx?id=7454
Gene Amplification by Polymerase
Chain Reaction (PCR)
Gene specific amplification
without purification
Amplification via temperature
cycling
Special Taq (Thermus
aquaticus) DNA polymerase
Detection of Helicobacter
pylori, Borrelia burgdorferi,
as well as hepatitis, HIV, and
West Nile virus
Restriction Enzyme Recognition Sites
Restrictive endonucleases
recognize 4-8 bp
sequences within the DNA
and cleave at a specific site
DNA strands that are cut in
a symmetric fashion are
palindromic
Blunt and Sticky DNA Cuts
Molecular Cloning by Recombinant
DNA Technology
• Cut by restriction
enzymes
• Anneal an uncatalyzed
reaction
• Re-close by DNA ligase
Screening for
Colonies Containing
the Plasmid with the
Insert
Size-Specific Cloning Vectors
Recombinant Protein Products
Generating a Restriction Map
Where are the
restriction sites
located on the 20
kb fragment that
would generate
this map?
.
.
.
Transcript Analysis via Microarray/DNA Chip
How to examine gene expression changes
with a given treatment
mRNA
↓
cDNA fluorecent labeling
↓
cDNA/oligonucleotide
Hybridization
↓
Slide scanning
↓
Data analysis
Differential Arabidopsis Gene Expression
with GB03 Exposure
RNA
Extraction
cDNA
Synthesis
Label
Hybridize
Wash
Scan
Block 8
Full Image
cDNA Synthesis and Labeling
O
HN
-
O
O
O
O P
O P
O P
O-
O-
O-
O
O
O
NH2
N
OH
Amino Allyl-dUTP
SO3-
O3S
N
N
O
O
O
O
Cy5 Dye
N
Microarray Data Analysis
Chapter 3 Problems:
1, 2, 7, 8, 9, 13, 15, 19, 21, 25, 29,
61, 62,