Griffith

(1928)

Transformation Principle

Studied Strep. Pneumoniae

S strain kills mice, R strain does not.

R+heat killed S; kills mice.

Hershey and Chase

(1952)

"Blender Experiment"

Bacteriophage is a virus that infects bacteria, only genetic material enters the cell. 32-P labeled DNA, 35-S labeled protein, only 32-P found in cells.

Chargraff

(1952)

Chargraff's Rules

A=T, G=C

Purines (A,G) = Pyrimidines (T, C)

A+T : G+C ratio is variable.

Watson and Crick

(1953)

Structure solution infers inheritable material.

Based on x-ray diffraction pattern by Franklin.

Double helix, bases in center.

Properties of DNA

-Nucleotides strung into nucleic acid by phosphodiester bond.

-Double Helix from H-bonding of bases.

-Base pairing rules; complementation.

-Denaturation/renaturation.

-Antiparallel, 5' → 3'

Restriction Enzymes

Cut DNA.

Cut sites are sequence specific and palindromic.

Overhangs can allow fragments to reanneal. (Sticky ends)

DNA ligase seals strands.

Gel Electrophoresis

Seperates DNA by size.

Agar slows down DNA. DNA is negatively charged so it moves towards positive charge.

Smaller bands migrate faster.

Differences in restriction fragment lengths provide useful information.

Southern Blot

Depends on hybridization (denaturation and renaturation).

If too many bands on a gel, certain bands of interest can be highlighted.

Probe binds desired sequence.

(Run gel, transfer DNA to blot, incubate with a probe, expose to film)

Probe

PCR

(polymerase chain reaction)

Uses a thermophilic DNA polymerase to make many copies of DNA.

Also need primers (like probes) that "flank" the region of amplification and free nucleotides.

Denaturation (94 C) seperates template DNA strands.

Annealing (60 C) primers bind template.

Polymerization (70 C) DNA polymerase makes new strands.

Each cycle doubles DNA, usually run 30 times.

DNA Sequencing

Modified "Sanger" Method.

Uses ddNTP (missing 3' OH) to arrest polymerization.

Each ddNTP is a different color.

Primer, template, polymerase, normal dNTPs, and small amount of labeled ddNTP.

Run modified gel electrophoresis with detection laser to read colors.

DNA Cloning

Steps

Remove gene of interest (GOI) using restriction enzymes or PCR.

Cut plasmid and GOI with same restriction enzyme and ligate.

Transform bacteria with recombinant plasmid.

Select bacterial clone containing plasmid with GOI.

DNA Cloning

Library Production

An extension of cloning where an entire set of genes can be stored.

Genomic Library Production

All DNA of organism is cloned.

Cut entire organisms DNA with restriction enzyme. Clone randomly into a vector (can use plasmid or phage). Transform into bacteria and store. Library can be probed to pull out GOI.

cDNA Library Production

Only expressed DNA is cloned.

Organism's mRNA is isolated (only expressed DNA (genes) will make mRNA).

Reverse transcriptase synthesizes cDNA from RNA.

cDNA is cloned into a vector and stored.

RT-PCR

DNA Microarrays

Allows global gene expression.

Combines Southern Blot with cDNA libraries.

DNA Microarrays

Method

Production of microarray chip. Location of each gene specified on grid. Single stranded cDNA or oligos to genes attached to glass. Sample production. Apply conditions to cells/tissues, isolate mRNA. Reverse transcribe labeled cDNA probes (one set green, one set red). Hybridization, Mix samples sets and incubate. Competitive binding to chip.

Colors show what binds. Computers read, Cluster analysis.

DNA Microarrays

Applications

Research- genes involved in a pathway, condition, or disease.

Drug Discovery- tested to repress gene expression.

Clinical- treatment plan can be assessed by expression profile (cancer).

Toxicology- screen for chemical effects.

Cloning Plants

Cloning Animals

Nuclear Transplantation

An egg cell (with its nucleus removed) becomes an acceptor for new nucleus.

(e.g. Dolly)

Stem Cells

Undefferentiated cells that can be fated to different cell types.

Embryotic stem cells can become any cell type.

Adult stem cells are limited, usually only to blood cells.

Applications of DNA Technology

Medical- Disease Diagnostics and Forensics (PCR fingerprints, restriction analysis, and sequencing can determine genetic diseases or unique sequences). Gene Therapy (vectors can deliver a normal gene to a patient). Production of Pharmaceutical Products (clone and express genes such as insulin).

Agriculture- disease resistance genes, antibiotics can be inserted into agricultural species.

Environmental Clean Up - bacteria have been altered to eat oil and die when oil runs out.

DNA Technology

Ethics

GMO foods- safety and environmental concerns, intellectual property rights.

Screening and Privacy Issues- discrimination (Sickle cell in military) versus reducing disease (Tay Sachs), government/insurance agencies.

Innaporpriate use- cloning humans, bioterrorism.

Human Genome Project

Government Method

Three step traditional way;

Genetic map to physical map (from libraries), to sequencing small fragments.

Human Genome Project

Craig Venter

Company: Celera

"Shotgun" approach;

Create a library and sequence randomly, use computers to piece together fragments by overlap.

Bioinformatics

Use of computational methods for analyzing biological data.

Sequence Information- studied for biological function.

Proteomics- studies proteins on a genome scale. Creates interaction maps, individual protein structures.

NCBI

National Center for Biotechnology Information

Allows DNA and protein searches.

Genbank- allows search of any sequence.

BLAST- can compare DNA or protein sequences with others for similarity.

Protein Chromatography

Seperation using a bead matrix.

Gel Filtration- size seperation

Ion- Exchange- charge seperation

Affinity- specific binding

Protein Electrophoresis

SDS-PAGE

(size seperation)

SDS unravels protein and coats with negative charge.

Then can use electrophoresis + western blot.

Antibodies

Defence Molecules

Have high antigen specificity.

Proteins injected into mice can induce antibody production specific for the protein.

Western Blotting

Similar to Southern Blotting but the probe used is an antibody.

Sensitivity can be greatly increased by using multiple layers of antibodies.

Immunoprecipitation (IP)

Specific molecules can be precipitated by antibody binding.

ChIP- chromatin can be used to find a specific gene that a protein is binding.

Immunohistochemistry

Determining Composition of a Protein

Sequencing- direct chemical degradation reactions.

Mass Spectrometry- proteins are broken down, ionized, and read by an analyzer.

Determening 3-D Structure of a Protein

X-ray Crystallography- Crystallize protein and hit with X-ray. Get diffraction pattern.

NMR (Nuclear Magentic Resonance)- Measure distance between atoms. Advantage: no need to crystallize protein.

Disadvantage: Can only do with small proteins <40kD

RNA Interference

A mechanism designed to destroy foreign or harmful RNA.

Anti-sence RNA sequences bind to mRNA.

Introduction of foreign RNA results in its destruction.

Introduction of double-stranded RNA results in destruction of all RNA with the same sequences.

RNA Interference

Applications

Research- Block gene expression; Can be done temporarily which is useful for lethal genes. Combined with genomics, can silence many genes at a time.

Therapeutics- Prevent spread of virus infection. Cancer (and other disease) causing genes can be silenced.