HG exam 3 pgs. 21-25
-2. Hydrogen peroxide (H2O2)
- 3. Peroxynitrite (OONO)
- Comes from = poor diet, smoking, alcohol, lifestyle, sun, too much exercise, too little exercise, fast food, air pollution, medication, pesticides.
- Just about everything we do results in producing free radicals
Glycation
- Glycation = _____non-enzymatic rxn_____________between reducing sugars and proteins/lipids/nucleic acids (macromolecules)
- Stiffness and loss of elasticity in arteries & organs
- Reduction of sugars causes accumulation of glycation end products in the extracellular matrix
- Protein molecules then crosslink sugar molecules = advanced glycation end products (AGEs)
- Body does not recognize as normal, makes antibodies to attack = ____inflammation__________
- Deposits into skin = ____wrinkling ______
Genetic technology: pcr & sequencing
PCR - what goes in
- Mimics what happens in your cells when they undergo DNA replication
- primers____ known sequence flanking area of interest
- ______DNA polymerase_________ enzyme required to add nucleotides to new strand
- template DNA________ what you want to amplify from
- ___nuceotides_________ to add on to the amplified strand
PCR thermocycler steps
- 1. Initial denaturation
- 2. Denaturation
- 3. Annealing
- 4. Extension
- 5. Final extension
- denaturation, annealing, and extension are repeated 30-40 cycles
PCR thermocycler steps
1. Denaturation
- Typically, performed at 94°C
- Allows for full denaturation of the template strands, especially long DNA strands (breaks ____hydrogen bonds________ between strands)
- Will be repeated each of the 30 - 40 cycles
PCR thermocycler steps
- 2. Annealing
- Typically, at 54°C
- Lower temperature allows primers to anneal__ to template
- Have 2 primers (forward & reverse) for each of the parent strands
- Will be repeated each of the 30-40 cycles
PCR thermocycler steps
- 3. Extension
- Typically, at 72°C
- Optimal for DNA polymerase to bind to primer/template
- Adds free nucleotides______ to new strand
- Will be repeated each of the 30-40 cycles
PCR thermocycler steps
- 5. Final Extension
- Typically at 72°C
- Makes sure all strands are completely finished
How to see your PCR
- Gel electrophoresis
- DNA is negatively ________charged
- If you apply an electrical current it will migrate (from = – to +)
- Use a chemical like ethidium bromide to visualize under UV
First Generation sequencing
- Sanger Sequencing (Aka.....chain termination method or dideoxynucleotide method)
- Uses only one strand of the double stranded DNA
- Start synthesizes new DNA strand using primer
- Uses dideoxynucleotides (ddNTPs = ddG, ddA, ddC, ddT) & regular nucleotides
- When a ddNTP is incorporated, ___elognation stops___________
- Results in numerous fragments with ddNTP on end
- Separate fragments on gel, align sequence and read it
Sanger sequencing
- How to read a Sanger Sequence
- Start at the top and work down the gel
- 1st use = 1977
- Used for decades, some still today
- Drawback = ___cost & time__________
Second generation sequencing
- Called “sequence by synthesis”
- Benefits over 1st Generation Sequencing:
- Millions of short reads in parallel
- Faster & low cost
- Don’t have to do gel electrophoresis
Second generation sequencing
- Fragment DNA
- Run PCR to add adenosine_________ to ends that primers attach to
- Add to flow cell that has adapters
- Primers bind to adapters
- Amplified and bends to match next adapter
- Fluorescently labeled nucleotides bind during amplification
- This gives a color signal to record the base
Third generation sequencing
- Called “single molecule real time read”
- Benefits over 1st Generation Sequencing
- 2 nd Generation requires an initial round of PCR first, 3rd Generation does not
- Complex genomes with lots of repeats confuse 2nd Generation, 3rd Generation does not
- 3rd Generation does not need chemical labeling
Third generation sequencing
- Nanopore
- _____single strand _____________DNA is fed thru a small pore opening (10-9 meters)
- Constant electrical field is generated
-Each nucleotide changes the current differently
Sequencing coming to a bedside near you
- Illumina $30,000 sequencer = human genome 1 hour = $1000
- Establish in hospitals to:
- Avoid cost of drug/therapy that has little chance of success
- Avoid/limit patient side effects
- Decrease disease burden looking for right treatment
- Act in preventative manner by predicting susceptibility
- Making correct diagnosis
- Monitoring and typing infections
Genomics - human genome project
- Goals (started 1990 by Watson, used WBC 2 males & 2 females, started 1990 & finished 2003) Publicly funded
- 1. ____identify_____ all human genes
- 2. ___map location_________ of all human genes
- 3. ____sequence________ all 24 chromosomes (looked at only euchromatin)
- 4. Analyze ___variation_______ between humans (ex. SNPs, VNTR)
- 5. Map & sequence genomes of _____model organisms _________ for future medical research
- 6. Develop new _____genetic technologies______________ to speed up research
- 7. Give findings to other scientists & ___general public__________
- 8. Set up ___ELSI__ program (Ethical -Legal- Social)
Genomics - human genome project
- How to sequence a genome
- Step 1 : Cut the genome into ______small overlapping pieces_________________
- Step 2 : Determine the ____sequence________ of smaller pieces
- Step 3 : Use ____algorithms________ to find overlaps
- Step 4 : overlap________ pieces to determine sequences
- * However, using this method missed CNV__
Sanger Sequencing
- Frederick Sanger, 1977
- Basically combines PCR___of unknown DNA segments and ___gel electropheresis_____________
- Uses _____fluorescently___labeled__________ dideoxynucleotides (lacks a ____3’ hydroxyl group ________)
Genomics - human genome project
- Gov’t funded:
- _____clone by clone method__________________
- _____1 chromosome @ a time__________________
- ________slower_______________
- ________doesn’t miss repeats_______________
Celera Genomics (Craig Venter)
- ___whole genome shotgun____________________
-_____entire genome at once_________________
- ______faster_________________
- _______misses repeats_______________
Genomics - human genome project
- The results:
- HG = 3.1 billion nucleotides
- Less than 2% of the HG is protein coding
- We are all 99.9% similar (SNP & CNV)
- Average size of a gene = 25kb
- 50% sequence similarity to other organisms
- Genes not uniformly distributed (Chromosome 1 most, Y chromosome least)
- Largest protein gene = Dystrophin (2.5Mb) (1Mb = 1,000,000 bp )
Genomics - human genome project
- Cooler ways to sequence came from the HGP $3 billion & 10 years → $1000 & few hours
- Chain termination - Fluorescent labeling
- Nanomaterial
Genomics - proteomics
- Proteomics is the identification, characterization, & quantitative analysis of the proteome of a cell, tissue or organism
- Which is larger: proteome or genome?
- Due to protein ____modification__________ & ______alternative splicing _____________
- Looks at :
- 1. Protein - protein interactions
- 2. Protein regulation
- 3. Protein modification
- 4. Protein location
- 5. Protein detection & quantization
- Methods
- 1. Isolate proteins from a _____single organelle__________________
- 2. Isolate entire protcome__________ Three areas of proteome analysis
- 1. ___expression__________ proteomics
- 2. ____bioinformatics___________ proteomics
- 3. ____functional___________ proteomics
Genomics - microbiome
- Human Microbiome Project 2007 - 2012 ($115 million)
- Sequenced the bacterial___, fungi__, & viruses__ that make up the microbiome of 250 healthy adults
- Sampled 17 areas on body and sequenced the 16s rRNA________ of bacteria
- Goals
- 1. Do humans share a core________ human microbiome?-
- 2. Does the microbiome ____change with health____________________
- 3. _____develop new technology__________________ to analyze the microbiome
- 4. Address ___ELSI____________
Genomics - microbiome
- Obtained 1000x more sequence data than the HGP
- 10,000 different bacterial species, 100 trillion
- Microbiome starts at birth
- We all have different microbiomes with similar groups
- More diverse = healthy
- Less diverse = unhealthy
- Smoking v pregnancy
- IBD & C. diff
- Twins
- Microbe fingerprint