Biotechnology in Clinical Cases Flashcards
Define Nucleic-Acids based Molecular diagnosis
Detects various pathogenic mutations in DNA and/or RNA samples
What is Sickle cell anemia?
- Genetic disease which is caused by a single nucleotide change in the 6th amino acid of the Beta-chain of hemoglobin
- Pauling introduced the term molecular disease based on discovery that SINGLE amino acid change leads to this disease
- Abnormal RBCs can cause clumps and block blood flow.
Do Human Red Blood cells have a nucleus?
NO! They go through nucleus ejection.
What did the following scientists accomplish:
- Watson and Crick:
- Perutz and Kendrew:
- Sanger:
- Milstein and Kohler:
- Watson and Crick: DNA structure
- Perutz and Kendrew: Protein sequence
- Sanger: Insulin and DNA sequencing
- Milstein and Kohler: Monoclonal Ab
Briefly explain X-Ray Crystallography
- Shining a beam of X-rays onto a crystal, causing the electrons surrounding the atoms in the crystal to diffract the X-rays in specific directions
- This creates a unique diffraction pattern that can be analyzed to determine the precise arrangement of atoms within the crystal, thus revealing the molecule’s 3D structure
What is the principle of PCR?
- Denaturation 94°C
- Annealing 55°C
- Forward and Reverse Primers - Extension 72°C
- Only dNTP’s
More generalized is
1. Temp. Cycling
2. Every cycle DNA between primers is duplicated
What components are involved in PCR?
- Template DNA
- Flanking Primers
- Thermostable polymerase (Taq polymerase)
- dNTP
- PCR Buffer (mg++)
- Thermocycler
Explain in detail how PCR occurs w/ Denaturation, Annealing, and Extension
- Denaturation
- Trade-off between denaturing DNA and NOT denaturing Taq polymerase @ 95°C
- Note: Taq 1/2 life = 40 min at 95°C, 10 min at 97.5°C - Annealing
- Trade-off between efficient annealing
- 2-5°C below Tm - Extension
- Temp. optimum for Taw polymerase
- 72°C
What is Huntington’s Disease
- Genetic disorder by abnormal body mvmt. and reduced mental abilities
- mutation in Huntingtin gene
- Non-HD individuals have <30 repeats of “CAG” trinucleotide
- HD-affected individuals have >36 “CAG” trinucleotide repeats
- PCR used to see if individual has HD by amplified DNA and sequencing
What is Human Immunodeficiency Virus (HIV)?
- Retrovirus that attacks immune system
- HIV tests rely on PCR with primers that will only amplify a section of the viral DNA found in an infected individual’s body fluids
- AKA, if there is a PCR product, person likely is HIV positive
What is SYBR GREEN used for?
- Labels DNA to fluoresce
- Also, relative quantitative
Define Droplet Digital PCR
- Exact copy number of DNA of the gene! QUANTITATIVE
- Accurate absolute quantification of template molecules by separation of target molecules and counting statistics
What are the 5 Goals of the Human Genome Project?
- To identify ALL of the genes in human DNA
- To determine the sequences of the 3 billion bases that make up human DNA
- To create databases
- To develop tools for data analysis
- To address the ethical, legal, and social issues that arise from genome research
Prospective biospecimen collection and retrospective case analysis will yield _______ _______?
Understand the flow chart involved!
Interconnected Results
Epigenetics –> Gene Regulation –> RNA and Protein Markers
Explain Microarray Platforms
- Based on idea of DNA complementary sequence hybridizations.
- Potentially detect and survey a large number of genes at once.
- Workflow: Reverse transcription, IVT with labeled nucleotides, array hybridization, staining, washing scanning.
- PROS/CONS: Rapid and streamlined protocols, standardized analysis; biased target collection, levels but limited sequence information
- Note: Higher fluorescence = Higher expression
- Analyzes thousands of genes in one experiment
- RELATIVE EXPRESSION!!!!
T/F: Gene expression can be used in the sense of turning on and off depending on epigenetic mechanisms
TRUE!
Note: roughly only 20% of genes are expressed at a specific time
Dideoxy (Sanger) Method includes what 4 steps!
- Denaturation
- Primer attachment and extension of bases
- Termination (insert dNTPs)
- Gel electrophoresis
Sequencing gels are read from (top to bottom OR bottom to top) aka (5’–> 3’ OR 3’–>5’)
Sequencing gels are read from BOTTOM TO TOP aka 5’–>3’
Sanger Sequencing is also known as
Sequence of Termination by Chain
Explain Shotgun Sequencing
- Large-scale sequencing requires DNA to be BROKEN INTO FRAGMENTS (Cutting with enzymes OR Shearing with mechanical forces)
- DNA is duplicated into a vector
- Individually sequenced
- Assembled electronically
Next Generation Sequencing (NGS) Platforms
- Ex.) Pyrosequencing Roche, Sequencing by Ligation Life Tech SOLiD, H+ Ion Generation Life Tech In Torrent, and Reversible Dye Terminators Illumina
- Attractive for clinical applications since individual sequencing assays were costly and laborious - serial “gene by gene” analysis
Explain Masssively Parallel Sequencing
see video!!!
Explain Illumina Flowcell Platform
see video!!!
Explain Ion Torrent Sequencing
- going through a nanopore with sensitive electronic detections they detect the differences of the protons carried by A,T,C, and G
- Dependent on semiconductor sensors without using optics or any sensors.
Q1-24 involves molecular imaging techniques!
Q1-24 involves molecular imaging techniques!
Who discovered X-rays?
Wilhelm Conrad Roentgen
How do X-Rays work?
- Electromagnetic Radiation
- They penetrate matter
- Cause fluorescence of some atoms
- Can expose films
- Can cause Biological Damage (aka mutations in DNA)
What is the X-Ray tube?
- Cathode is the electron source
- Tungsten filament
- Negatively charged electrons charged concave cup around filament
- Focal spot
- Thermionic emission: current applied wire heats up and electrons escape
- Anode is the target which electrons strike
- Tungsten target
- Stationary: Anode in large block of copper aka lots of heat. Used in portable units
- Rotating: Disc rotates which spreads electrons around the target thus less heat build up.
What are 2 problems which exist with conventional radiography techniques?
- Inability to distinguish soft body tissue because of limited contrast (ex. blood-muscle); this can be fixed by the use of liquid contrast medium which has to be injected
- Inability to resolve spatially structures (aka we only see a 2D image) along the X-ray propagation axis resulting in loss of depth information (flat picture), because the 3D body is projected onto a 2D receptor
Explain Computed Tomography (CT)
- Uses X-rays to produce an image
- Cross-sectional imaging
- No superimposition of structures
- Requires computer manipulation of images
What are the detectors used in CT scans?
- Scintillation photodiodes
- Convert light into electronic signal
- Approx. 90% of the x-rays incident on the detectors are absorbed and contribute to output signal
- Used to record photon activity
What are CT scans good for?
- Demonstrates Abnormalities
- Metastatic lesions, aneurysms, abscesses, and fluid collections from infection or trauma - Interventional procedures
- Abscess drainage, tissue biopsy, and cyst aspiration
Explain CT Angiography (CTA)
+
List Advantages over conventional Angio
- Uses 3D imaging techniques to evaluate the vascular system
- Advantages over conventional angio:
- Image reconstruction without the use of more patient exposure to radiation or IV contrast
- Overlying structures can be eliminated
- Does NOT often require an arterial pressure
Define Contrast Media
- Used in most CT imaging to distinguish normal anatomy from pathology
- Iodine-based IV contrast
- Oral/Rectal contrast
Ultrasound Ex.) Bats
If a bat hears an echo 0.01 second after it makes a chirp, how far away is the object?
Distance = speed * time
(Speed of sound in air = 330 ms^-1)
Distance = 330 ms^-1 * 0.01 = 3.3 meters
Note: This is back and forth, so divide by 2 = 1.65m directly in front of the bat.
Diagnostic Imaging: Ultrasound
Brief Explanation
- High frequency sound is transmitted into the body, usually in very short pulses
- Returning echoes are analyzed to create an image
- Lower density = lower bounce-off
What is the principle of Ultrasound Imaging
- Short ultrasound pulses into tissue used
- Listen for sound reflected and scattered by tissues
- Display gray-scale image w/ brightness level is based on the echo strength
Are Ultrasounds safe?
- Mostly safe, there is NO radiation
- Ultrasound does generate a bit of heat
- 2D ultrasound is thought to be completely safe
- 4D is new, causes a bit more heat than 2D, but considered mostly safe
Explain how Ultrasounds resemble Doppler Effect
- Ultrasound is a wave
- If source moves towards the listener, wavelength gets shorter and frequency gets higher. The sound has a higher pitch
- Used to workout how fast blood cells move
Capabilities and Limitations of Ultrasound
Positives:
- Provides real-time imaging
- Non-invasive
- Essentially nontoxic
- Inexpensive
Negatives:
- Poor or no imaging through bones or gas
- Operator-dependent imaging quality
- Images more difficult to interpret than CT
Understand Diagnostic Imaging: MRI
- Uses a strong magnetic field and radiofrequency waves to image structures
- No ionizing radiation
- Hydrogen protons - water
- Cross-sectional imaging
- Great for soft tissue
Explain the specifics of MRI
(LONG ANSWER)
- Radio frequency is passed through the coils that excites the hydrogen protons in the target area
- The graident magnets are then activated in the main magnet and alter the magnetic field in the area that is being scanned, causing the hydrogen protons to spin
- The magnetic field runs down the center of the patient, causing the slowing hydrogen protons to line-up
- The protons either align themselves pointed towards the head or the feet of the patient, and most cancel each other out
- The protons that are not cancelled create a signal and are the ones responsible for the image
- The contrast dye is what makes the target area stand out and show any irregularities that are present
- The dye blocks the X-Ray photons from reaching the film, showing different densities in the tissue
- The tissue is classified as normal or abnormal based on its response to the magnetic field
fMRI has the capability to measure parameters related to several neural physiological functions including:
- Changes in phosphorus metabolism and metabolic byproducts
- Blood flow
- Blood volume
- Blood oxygenation
Define PET scan
- Nuclear medical imaging technique which produces 3D images of FUNCTIONAL PROCESSES in the body
- Detects metabolically active cells (i.e cancer)
How does a PET scan work (STEPS)
- Radioactive tracer isotope injected in blood circulation which is incorporated to biologically active molecule
- Waiting period while active molecule becomes concentrated in tissue of interest
- Positron emission decay (positron beta decay), emits a positron
- Positron travels a bit and finds electron
- The encounter annihilates them both, producing pair of gamma photon moving in opposite direction
- These are detected when reach scintilator creating burst of light detected by photomultiplier tubes
- Create image to find what is overreactive
What tracers are used in PET scans
Isotopes of carbon, nitrogen, oxygen, gallium, and 18F (temporary) instead of hydrogen
List SOME primary applications of PET scan
- Detect cancer
- Whether cancer has spread
- Determine blood flow to heart muscle
- Determine effects of a heart attack
- Map normal human brain and heart function
- Evaluate brain abnormalities like tumors and seizures
What are some benefits of combined PET/CT scanner
- Detect structure and function simultaneously
- Greater detail with higher level accuracy
- Greater convenience for the patient who undergoes two exams at one sitting rather than two different times
