A.
Module 44: Discovery of Genetic Material a.
Griffith (1928) - Transformation Principle (Fig. 2) i.
Transformation of Steptococcus pneumoniae . ii.
S strain kills mice, R strain does not kill. R + heat-killed S = killed. The R strain was transformed into an S strain. b.
Avery (1944) – proved that transformation was caused by DNA (Fig. 3) i.
Addition of protease or RNase still gave transformation. DNase prevented transformation. c.
Hershey and Chase (1952) and “Blender Experiment” (Fig. 4, 5) i.
Bacteriophage is a virus that infects bacteria. Only genetic material enters cell. ii.
32
P-labeled DNA,
35
S-labeled protein. Only
32
P found in cells. d.
Chargaff (1952), Chargaff’s Rules i.
A=T, G=C ii.
Purines (A,G) = pyrimidines (T,C) iii.
A+T to G+C ratio is variable e.
Watson and Crick (1953)-structure solution infers inheritable material. i.
Double helix: bases pair in the center, backbones on the outside. ii.
X-ray diffraction pattern by Franklin (Fig. 7)
B.
Review of Properties of DNA (from Module 12) a.
Nucleotides strung into nucleic acid by phosphodiester bond b.
Double helix from H-bonding of bases c.
Base pairing rules – complementation d.
Denaturation/renaturation e.
Antiparallel, 5’ 
3’
A.
Module 57: DNA Cloning a.
Basic steps of DNA cloning (Fig. 4) i.
Cut a plasmid and a gene of interest (GOI). Insert gene of interest into plasmid. ii.
Transform into bacteria. Select bacterial clone containing plasmid with GOI. iii.
Bacteria can be used to make a product or transfer GOI to another organism. b.
Restriction enzymes in cloning (Fig. 1, 2) i.
Cut sites are sequence specific and palindromic: e.g. Eco RI cuts G↓AATTC ii.
Overhangs (sticky ends) can allow fragments to specifically reanneal. Some enzymes can create blunt ends: e.g. SmaI cuts CCC↓GGG. iii.
DNA ligase seals strands c.
Library Production i.
An extension of cloning where an entire set of genes can be stored (Fig. 5). ii.
Genomic – all DNA of organism is cloned.
1.
Cut entire organism’s DNA with a restriction enzyme.
2.
Clone randomly into a vector (can use plasmid or phage).
3.
Transform into bacteria and store
4.
Library can be probed to pull out GOI (Fig. 7) iii.
cDNA – only expressed DNA (Fig. 6)

1.
Organism’s mRNA is isolated (only expressed DNA[genes] will make mRNA
2.
Reverse transcriptase synthesizes cDNA from RNA.
3.
cDNA is cloned into a vector and stored. d.
PCR (polymerase chain reaction) – (Fig. 9) i.
Uses a thermophilic DNA polymerase to make many copies of DNA ii.
Also need primers (like probes) that “flank” the region of amplification and free nucleotides. iii.
Each cycle
1.
Denaturation (e.g. 94 o
C) – separates template DNA strands
2.
Annealing (e.g. 60 o
C) – primers bind template
3.
Polymerization (e.g. 70 o
C) – DNA polymerase makes new strands iv.
Each cycle doubles amount of DNA. Usually run 30 cycles.
B.
Module 58: DNA Technologies a.
Gel electrophoresis separates DNA by size (Fig. 1, 2) i.
Agar (the gel) slows down DNA. ii.
A current is applied across the gel. iii.
DNA is negatively charged so it moves towards positive charge. iv.
Smaller bands migrate faster, larger fragments slower. v.
The 3-D structure of the DNA can affect migration (Fig. 3) vi.
Differences in band lengths provide useful information (Fig. 4) b.
DNA sequencing – Modified “Sanger” method (Fig. 7) i.
Uses ddNTPs (Fig. 6)
1.
These are dNTP with missing 3’OH. This arrests polymerization.
2.
Each ddNTP is labeled a different color ii.
Primer, template, polymerase, normal dNTPs. Each tube has a small amount of labeled ddNTP. This gives fragments of varying length. iii.
Run modified gel electrophoresis with detection laser to read colors. c.
Studying Gene Expression i.
RT-PCR – good for single genes (Fig. 8, 9)
1.
Reverse transcription step allows PCR analysis of only expressed genes and their levels.
2.
PCR is done normally after RT step ii.
DNA microarrays allows global gene expression (Fig. 11, 12)
1.
Combines southern blot technology with cDNA libraries
2.
Method: (Read Friend and Stoughton, 2002 on protected area of my website) a.
Production of microarray chip. Location of each gene specified on grid. Single stranded cDNA or oligos to genes attached to glass. b.
Sample production. Apply conditions to cells/tissue. Isolate mRNA. Reverse transcribe labeled cDNA probes (one set green, other set red) c.
Hybridization. Mix sample sets and incubate. Competitive binding to chip. d.
Analysis. Colors represent what binds: red, green, yellow, and black. Computers read. Cluster analysis.
3.
Applications a.
Research – genes involved in a pathway or condition or disease. b.
Drug discovery – drugs can be tested to repress gene expression. c.
Clinical – treatment plan can be assessed by expression profile.

d.
Toxicology – screen for chemical effects. d.
RNA Interference (Read Lau and Bartel, 2003 at protected area) i.
Overview
1.
A mechanism designed to destroy foreign or harmful RNA
2.
Anti-sense RNA sequences bind mRNA
3.
Discovery a.
Introduction of foreign RNA results in its destruction. b.
Introduction of double-stranded RNA results in the destruction of all RNA with the same sequences. ii.
Mechanism
1.
dsRNA is recognized by Dicer, a enzyme that cleaves the dsRNA into
22bp segments called siRNA (small interfering)
2.
siRNA is recognized by RISC (RNA induced silencing complex), a large protein complex. siRNA is unwound and one of the strands remains in
RISC
3.
The RISC-ssRNA complex can bind mRNA with the complementary
RNA sequence.
4.
Complementary RNA sequences are destroyed: a.
Slicer is an enzyme that cleaves b.
Ribosomes are blocked from translation iii.
Applications
1.
Research a.
Block gene expression. Can be done temporally which is useful for lethal genes. b.
Combined with genomics, can silence many genes at a time, similarly to microarrays
2.
Therapeutics a.
Prevent spread of virus infection (HIV, polio, hepC etc.) b.
Block cancer – cancer causing genes can be silenced c.
Any disorder that requires gene silencing
C.
Module 59: Organismal Cloning a.
Cloning Plants – Single Cell Cultures (Fig. 1) i.
Plants cells are totipotent: they can undifferentiate and redifferentiate. ii.
Any cell can be cultured and grown up to an adult plant b.
Cloning Animals – Nuclear Transplantation (Fig. 2, 3) i.
An egg becomes acceptor of a new nucleus. ii.
Egg nucleus is first removed. iii.
Donor nucleus is inserted. The best are from less differentiated cell types.
1.
Embryonic DNA is less modified.
2.
Development modifies DNA such as methylation. This decreases cloning success (Fig. 5) c.
Stem cells – are undifferentiated cells that can be fated to different cell types. (Fig. 6) i.
Embryonic stem cells can become any cell type. ii.
Adult stem cells are limited: usually only to blood cells.
D.
Module 60: Applications of DNA Technology a.
Medical i.
Disease diagnosis and forensics – PCR fingerprints, restriction analysis, and sequencing can determine genetic diseases or unique sequences. ii.
Gene therapy – vectors can deliver a normal gene to patient (Fig. 2) iii.
Production of pharmaceutical products – clone and express genes. GFP can be used as a tag for tracking a cloned gene (Fig. 4)

b.
Environmental cleanup – bacteria have been altered to be able to eat oil and die when oil runs out. c.
Improvement of agriculture (Fig. 7) – disease resistance genes, antibiotics can be inserted into agricultural species. d.
Ethics i.
Genetically modified products
1.
Safety of GM foods and environmental concerns.
2.
Intellectual property – ownership rights. ii.
Screening and privacy issues
1.
Sickle cell screening caused discrimination in military.
2.
Knowledge by agencies: government and insurance companies
3.
Tay-Sachs screening helped reduce disease. iii.
Inappropriate use
1.
Cloning humans.
2.
Bioterrorism – making anthrax spores, releasing viruses.
A.
Module 61: Human Genome Project a.
Government (1990) vs. Craig Venter (1998). Both finished rough drafts in 2001. b.
Government used three step traditional way. Genetic map

physical map (from libraries)

sequence small pieces. (Fig. 2) c.
Venter used “shotgun” approach. Create a library and sequence randomly. Use computers to piece together fragments by overlap. (Fig. 4)
B.
Bioinformatics – use of computational methods for analyzing biological data a.
Sequence information is studied for biological function b.
Resources: NCBI – Nat. Center for Biotechnology Information. Allows DNA and protein searches. i.
Genbank – a large database that allows search of any DNA sequence. ii.
BLAST – can compare DNA or protein sequences with others for similarity.
(Fig. 5) iii.
Gene ontology attempts to provide standardized language to describe and compare gene function (Fig. 6) c.
Studying systems – many genes at once. i.
Proteomics – studies proteins on a genome scale ii.
Interaction maps can be assembled (e.g. all interactions related to a disease or a process) (Fig. 7) iii.
Transcriptomes describe transcription on a global level
C.
Module 62: Genome Diversity a.
Comparison of genomes – number of genes and spacing contribute to complexity (Tab. 1,
2, 3) b.
Humans make many more proteins than number of genes. This is due to alternative splicing (Fig. 1, 2) c.
A large portion of human genome is non-coding (Fig. 3) d.
Phylogenic trees (evolutionary trees) help describe relationships among organisms or genes. e.
Gene alignments can help in understanding origin of a virus, e.g. HIV
A.
Chromatography separates using a bead matrix (Panel 4-4)

a.
Gel filtration – size separation b.
Ion-exchange – charge c.
Affinity – specific binding
B.
Electrophoresis (Panel 4-5) a.
SDS-PAGE – size separation
C.
Use of antibodies (Panel 4-6) a.
Overview i.
Antibodies are defense molecules ii.
They have high antigen specificity iii.
Proteins injected into mice can induce antibody production specific for the protein b.
Affinity chromatography – protein binds to antibodies attached to beads. c.
Western Blotting – tagged antibodies probe an SDS-PAGE. (Module 50 Fig. 13) d.
Immunoprecipitation (IP) – specific molecules can be precipitated by antibody binding. i.
ChIP – chromatin can be isolated to find a specific gene that a protein is binding
(Module 49 Fig. 2) e.
Immunohistochemistry – tissues can be sectioned and probed by antibody to highlight parts of cells.
D.
Determining composition a.
Sequencing – direct chemical degradation reactions b.
Mass spectrometry – proteins are broken down, ionized, and read by an analyzer.
E.
Determining 3-D structure a.
X-ray crystallography. Crystallize protein and hit with X-ray. Get diffraction pattern.
(Module 10 Fig. 9) b.
NMR-magnetic field. Measure distance between atoms. Advantage: no crystallization.
Disadvantage: limited to 40kD