HG exam 21-30
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
Microbiome - brain/gut axis
- Brain - gut axis
- 1.__lymphocytes___________ sense gut lumen, release cytokines
- 2. _____vagus nerve________ terminals activated by gut microbial peptides
- 3. ____neurotransmitters_______________ made in response to microbial metabolites
- 4. hormone______ release via hypothalamic pituitary, modulates gut composition
- 5.__neural activation______________– sympathetic activation produce neurotransmitters modify microbiota composition
- How do we study the BGA?
- 1. ___germ-free mice_____________ = mice born devoid of any normal bacterial flora
- 2. ___probiotic_____________ = Lactobacillus & Bifidobacterium reduce anxiety levels, lower inflammatory cytokines, reduced depression
- 3. _____antibiotic intervention___________________ = reduce biodiversity, delays re - colonization
- by probiotics, expression of GI symptoms
- 4. infection____ = increases inflammation, anxiety, Campylobacter pathogen activated brain regions associated with processing GI sensory information
Genomics - metagenomics
- Metagenomics is also called ___environmental genomics______________ and uses the ____whole genome shotgun____________approach to sequence the genomes of entire communities of microbes in environmental samples of water, air, soil.
- Want to understand
- 1. ___interactions________ of microbial communities & environment
- 2. ___classify________ new species
- 3. Identify genes with novel functions__________ (medicine/biotech)
- 4. Be able to isolate DNA directly from ____sampled area___________
- The Sorcerer II Global Sampling Expedition (Craig Venter)
Genomics - other “omics”
- ___metabolomics__________ : Study of proteins & enzymatic pathways in cell metabolism
- gkycomics__________ : Study of carbohydrates of cells or tissue
- ____toxicogenomics__________: Study of the effects of toxic chemicals on genes
- ___transcribtomics___________ : Study of all gene transcripts in tissue or cells
- _____stone age genomics____________: Study of ancient DNA (oldest 70,000)
- ___nutrigenomics______________ : Study of the interactions of diet and genes
Genomics - systems biology
- Is an intergrative__________ process
- Interprets ____genomic information_________________ in the context of _____biological pathways___________
- ____interactome___________ is the
- 1. Protein to protein interactions
- 2. Protein to nucleic acid interactions
- 3. Protein to metabolite interactions
- ___network map___________ – shows interacting proteins, genes, & molecules, but cannot tell you when & where the interaction occurs
- Important for:
- 1. Scientists – model potential interactions in normal and disease processes
- 2. Pharmaceuticals – drug discovery, development, detection
Genomics - bioinformatics
- Bioinformatics = the use of _____computers & mathematics _______________to organize, share, and analyze data related to gene and protein sequence____, structure, function & expression
- ____databases_______ are essential for the storage and sharing of all this newfound information
- gen bank____ is the largest publicly available database of DNA sequences (doubles in size every 14 months, 100,000 different species)
- BLAST will tell you
- 1. Genes with similar sequences
- 2. E - values = matching the sequence based on chance
- 3. Identities = similarity score based on alignment
- 4. Gaps = deletions or insertion points
Pharmacogenetics & personalized medicine
The ACCE test
- A = ___analytical_______________= how well does the test measure what it claims to measure (accuracy)
- C = ___clinical valibdation_____________= how well does the test predict the health outcome that it claims
- C = _____clinical utility__________= how useful are the results to the patient
- E = ____ethical aspects___________ = is it voluntary, insurance, employment
Pharmacy meets genomics
- First step to personalized medicine = personalized prescribing
- Medicine has different effects on different patients
- Some patients have different sensitivities_______ = higher/lower dose
- Some individual patients have no__ therapeutic effect
- Some individual patients have ___adverse____ reactions
- Some patients cannot take a drug due to ____interactions________ with another drug
MTHFR genetics
- Considerable evidence of folic acid and diseases like depression, bipolar disorder
- MTHFR gene mutations = can’t convert folate to biologically active form Methylfolate
- Methylfolate essential for making ___neurotransmitters___________ serotonin & dopamine
- This can both cause depression AND cause you to be resistant to antidepressive treatments
- No Methylfolate = cannot add methyl___ groups
- Due to SNP in one of two areas of MTHFR gene (50% of population has at least 1 SNP)
- The more SNPs mean the greater it :
- Reduces response to antidepressants
- Reduces response to chemotherapy
- Increases adverse reactions to nitrous oxide (dental anesthesia)
Terms
- pharmogenetics_____________ = study of the roles of specific genes in drug interactions
- ___charmogenetics______________ = uses genome - wide studies to study drug interactions
- ___pharmokinetics_______________ = studies absorption, activation, metabolism, excretion of drugs ( what the body does to the drug)
- ____pharmodynamics______________ = studies the actual target response (what the drug does to the body)
How genetics play a role in medical drugs
- Genetic factors are all part of the different pharmacy + genetics field
- 1) ___absorption_________ = differences in how patients differ in ability to transport an oral drug to
bloodstream
- 2) ___acrivation_________ = drugs that are given in the form of a prodrug that have to undergo
enzymatic reactions (by the liver) to become active
- 3)____target response____________= differences in how patients process or pathway that is targeted by
the drug response due to local concentration
- 4) _____catobolism and secretion_________________= differences in how patients break down & dispose of the
drug (slow metabolizers have longer/stronger response to drug that fast metabolizers)
- Adverse drug reaction in US = 100,000 deaths/year
- Wasted time in treatment, wasted money, extra suffering
How drugs are metabolized & genetics
- Phase 1 reactions = produce the ____biologically active_______________ molecule
- Polymorphic variations in enzymes among people alter these reactions
- CYP2D6 enzyme= used in metabolism of 25% of all drugs
- poor, intermediate, extensive, ultra rapid metabolizers
- poor = codeine is ineffective______
- ultra rapid = codeine has ____increased risk_________of sedation/impaired breathing
- also linked to beta blockers for hypertension & antidepressants
How drugs are metabolized & genetics
- Phase 2 reactions = produce water soluble molecule for ___excertion________
- Polymorphic variations in enzymes among people alter these reactions
- Thiopurine methyltransferase (TPMT):
-transfers methyl group to immunosuppressant drug azathioprine
- ___heterozygotes___________ = normal elimination of the drug
- ___homozygotes___________ = life threatening bone marrow toxicity due to poor elimination
Where pharmaceutical companies come in
- Want to make $ by selling drug to widest possible range of patients: Pharmaceutical genetic medicine: 15 years, $1 billion
- Merk 2004 Vioxx anti-inflammatory for arthritis increased heart attack/stroke
- Stages of development:
- ___preclinical__________= lab studies in vitro & animal = explores toxicity & pharmacokinetics
- _____microdosing__________ = single small dose given to 10 - 15 healthy volunteers (first to involve people)
- explores preliminary pharmacodynamics & pharmacokinetics
- Phase 1 = 100 healthy volunteers
- explores general safety, tolerability, goal how to _____deliver_____ (pill, injection, etc ) & what is the highest dose
- Phase 2 = 100’s of patients
- explores general safety, tolerability , survival time, _____quaility of life_________
- Phase 3 = 1000’s multi - centers & Worldwide, randomized 10,000+ patients Assigned to 2 groups: 1) current treatment vs 2) new treatment ___double blind__________ so participant & research team don’t know who gets what treatment Last step before ___FDA approval_______
- Phase 4 = Post market _____surveillance___________ (largest group: 100,000+ patients)
- explores adverse reactions, long term health implications & off label uses
Genetic medicine - treatment - pharmacogenetics
- Typically work in 1 of 3 ways
- 1. Enzyme ___replacment_________ therapy = recombinant human enzyme infused to
- compensate for deficient enzyme (LSD – no enzyme to breakdown, must infuse, $500,000 year)
- 2. Substrate reduction_______therapy = oral drug that reduces the level of substrate so that the enzyme works more efficiently
- 3. Pharmacological ___chaperone_________therapy = oral drug that binds misfolded protein so that the protein function is restored
- Lysosomal storage disease example (Gaucher disease)
- Antibiotics (aminoglycosides) – works on bacteria by shutting down ____protein synthesis__________ , but also affect nonsense mutation____________ in humans by distorting the ribosome so that protein synthesis to continue. Currently, about 12%_ of all mutations are nonsense
A closer look at cystic fibrosis & treatment based on genotype
- CFTR gene
- 2000 known mutation
- F508___deletion
- 90% of CF patients (27,000)
- October 2019 FDA approved Trikafta
Expression profile of tumors & treatment
- Traditional approach classify stage________:
- tumor size
- involvement of lymph nodes
- presence of metastases
- Personalized medicine approach _____gene expression_________:
- Microarray to analyze tumor RNA
- Identify genes that are up/downregulated
- + stage cancer (early vs late)
- + good or poor survival outcome
- + treatment tailoring
Success of personalized medicine: biomarkers
- predisposition __________ biomarkers = genetic susceptibility.
- How likely is a person is going to develop a health disorder if they have a certain biomarker.
- Example: BRCA1&2 indicates an increased susceptibility to breast cancer (family history)
- Guides persons future medical care
- diagnostic________ Biomarkers = genetic confirmation.
- Does a patient have a certain disorder.
- Example: CFTR. Presence of mutation would indicate someone has cystic fibrosis.
- Earlier detection than physical examination or symptoms
- prognostic_______ Biomarkers = Genetic progression.
- How a disease may develop in an individual when they have been diagnosed.
- Oncotyping looks at 21 genes to determine likelihood breast cancer will come after initial treatment
- ___predicitve _______ Biomarkers = Genetic treatment.
- Helps to determine what treatments will work best in a particular patient with the least side effects
Other examples of personalized medicine - the need to screen for genetics
- HIV – Abacavir is a highly effective treatment for HIV, but 8% of patients suffer severe side effects.
- HLA - B 5701 gene variant causes ___hypersensitivity______________to the drug
- Can now screen patients for the variant & give an alternate treatment
- Rheumatoid arthritis – Azathioprine is an immunosuppressant used
- Some patients can’t break down drug, builds up in bone marrow killing immune cells____
- Variant of TPMT gene causes this, can now screen patients for the variant
- Pulmonary embolisms – Warfarin given to reduce blood clots______
- Must give at correct dose = too little no effect (blood clots), too much excess bleeding
- VKORC1 gene variation affects a patient’s sensitivity to Warfarin
Hurdles to personalized medicine
- Regulatory oversite – FDA So far there is a lack of standards that have not been addressed
- Intellectual property rights US Supreme Court – naturally occurring genes cannot be patented
- Reimbursement How & when to be charged, who pays
- Privacy & confidentiality
- Availability of tests Right now, so many conditions do not have an approved test
- No current bedside test