Answers in Studying Molecular Biology How does human genetic information differ from our closest...
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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
Are humans genetically programmed to contract certain diseases?
Answers in Studying Molecular Biology
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
Healthy Lung
Cystic Fibrosis LungCystic 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 Deoxy-nucleotides (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 can re-nature with single strands re-forming a double helix if cooling occurs slowly.
DNA Re- and De-naturation
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/FunctionsOxidation/Reduction Reactions
Other Nucleotides Molecules/FunctionsOxidation/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 Deoxy-nucleotides (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
Blunt and Sticky DNA Cuts
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
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
mRNA↓
cDNA fluorecent labeling↓
cDNA/oligonucleotideHybridization
↓Slide scanning
↓Data analysis
How to examine gene expression changes with a given treatment
Differential Arabidopsis Gene Expression with GB03 Exposure
Full Image
Block 8
RNA Extraction
cDNA Synthesis
Label
Hybridize
Wash
Scan
HN
O
NO
OH
O
O
PO
O-
O
NH2
O
PO
O-
O
P
O-
-O
NN
SO3-
O
ON
O
O
-O3S
cDNA Synthesis and Labeling
Amino Allyl-dUTP
Cy5 Dye
Microarray Data Analysis
Chapter 3 Problems:
1, 2, 7, 8, 9, 13, 15, 19, 21, 25, 29, 61, 62,