MI Semester 1 Flashcards

Label red peaks as

thymine (T).

Label green peaks

as adenine (A).

Label blue peaks

as cytosine (C)

Label black peaks

as guanine (G)

Label ambiguous areas where nucleotide peaks overlap

with an N.

Skips sections that contain…

Lots of N’s

What is the purpose of DNA Sequencing?

It’s how scientists determine the precise orders of nucleotides within a DNA molecule.

Cycle sequencing

modification of the traditional Sanger sequencing method. The components are DNA, primer, heat resistant DNA polymerase, 4 4 ddNTPs, fluorescently labelled with four different dyes and buffer containing Mg⁺⁺ and K⁺. It produces a set of DNA fragments that differ in length from each other by a single base. The fragments are then seperated by size and the basis at the end are identified, recreating the original sequence of the DNA.

What needs to be done before cycle sequencing?

Isolating the DNA from the sample. The DNA must then be processed so we can ensure we’re only working with DNA that we want to analyze.

How is DNA isolated?

DNA is isolated through performing Polymerase Chain Reaction. (PCR. )PCR produces many copies of the same DNA sequence.

What are primers?

Primers are short segments of DNA that attach to genes found in bacteria and viruses, so PCR makes multiple copies of the sequence, it is only copying a segment of DNA that belongs to the bacteria or virus present in Sue’s sample.

What is the first step of cycle sequencing?

The first step is to denature the stand of DNA, meaning it’s seperated into two strands. We call the respective strands the template strand and the complementary strand.

What is the template strand mixed with after the initial denaturing of the DNA?

It is mixed with primers, free nueclotides that are florescently tagged, (A, T, C, and G) , DNA polymerase, (enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to create two identical DNA strands from one original DNA molecule.)

The creation of each DNA fragment….

Starts with a primer. After the primer attaches to the DNA template strand, the DNA polymerase goes to work and adds complementary nucleotides to build the DNA.

Whenever a florescently tagged nucleotide gets added to the template strand…….

The DNA polymerase stops, and that chain is complete.

When does the cycle sequencing reaction stop?

When nucleotides run out.

What is the overall result of DNA sequencing?

The overall result of the DNA sequencing is DNA strands with different lengths that end with a floresecntly tagged nucleotide.

What do you do after you have all the fragments?

You seperate them by size.

What does the automatic sequencer do?

Performs gel electrophoresis to seperate the DNA by size, and then analyzes the results.

What do the thin capilary tubes inside the sequencer do?

These are where the floresecntly tagged DNA pieces will go. In these tubes, an electrical current is applied so negatively charged DNA will move downards to the postively charged end of the tube. (the bottom). These tubes are also filled with a buffer solution.

Which segments will move down the cappilary tube the fastest?

The shortest ones, so they reach the bottom of the tube the fastest. Then a laser beam will pass through the tube, making the nucleotides glow. The laser is intergrated with a computer that labels the DNA bases. The computer represents guanine as black because yellow is hard to see.

What does BLAST stand for and what does it do?

Basic Local Alignment Search Tool (BLAST)The program compares nucleotide or protein sequences and calculates the statistical significance of matches.

e-value

Short for Expect Value. The number of BLAST hits you would expect to see by chance. The smaller the number, the more likely the match is accurate and not by chance. If the E-Value is 0, there is zero chance that the sequence was matched with the query sequence at random. 

max identity 

Displayed as Ident on BLAST. The Max Identity value describes the percentage of nucleotides from the sequence that match with the query sequence. If the Max Identity is 100%, all of the nucleotides in the sequence match the query. 

1. Why is PCR used in the process of DNA sequencing? 

Polymerase Chain Reaction (PCR) is used in DNA sequencing to amplify specific DNA regions of interest. This amplification ensures that there is enough DNA material to sequence accurately. Without PCR, the amount of DNA in a sample might be too low for reliable sequencing. PCR also allows for the selective targeting of specific genes or regions, which is particularly useful when analyzing pathogens or specific mutations.

1. How can DNA sequencing be used to identify other classes of pathogens, such as viruses? 

DNA sequencing can identify pathogens by analyzing their genetic material. In the case of viruses, sequencing can detect and identify viral DNA or RNA. For RNA viruses, reverse transcription is used to convert RNA into complementary DNA (cDNA) before sequencing. Comparing the sequenced genetic material against reference databases allows scientists to determine the pathogen's identity, subtype, and possible mutations, which is crucial for diagnosis and treatment planning.

1. Explain how sequence data and information about patient symptoms led you to diagnose Sue’s illness. 

Sequence data can identify the specific pathogen or genetic marker associated with a disease. For Sue, DNA sequencing of her sample would reveal the genetic material of a pathogen (e.g., bacterial, viral, or fungal) or a genetic mutation. Combining this data with her symptoms (e.g., fever, rash, or respiratory distress) allows for a precise diagnosis. For example, if sequencing detects Borrelia burgdorferi DNA and Sue has a bullseye rash, this suggests Lyme disease. The integration of molecular and clinical data ensures accurate diagnosis and effective treatment.

1. How can DNA sequencing be used to identify genetic risk for certain diseases and disorders?

DNA sequencing can identify mutations or variations in a person's genome that are associated with genetic risks for diseases. By analyzing genes known to be linked to specific conditions (e.g., BRCA1/BRCA2 for breast cancer or APOE for Alzheimer’s disease), sequencing can reveal inherited predispositions. This information helps in assessing the risk level, enabling early interventions, lifestyle modifications, and personalized medical care to prevent or mitigate the disease's impact.

Outbreak

A sudden rise in the incidence of a disease

Pathogen

A specific causative agent of disease

Antibody

A protein secreted by plasma cells (differentiated B cells) that binds to a particular antigen and marks it for elimination.

Concentration

The amount of a specified substance in a unit amount of another substance.

Solvent

A substance, usually a liquid, capable of dissolving another substance

Solute

A substance dissolved in another substance

Solution

A homogeneous mixture of two or more substances, which may be solids, liquids, gases, or a combination of these.

How do you create a standard curve(use ELISA as an example)

To make a standard curve in ELISA, prepare a series of solutions with known concentrations of the target analyte. Perform the ELISA on these solutions and measure their signals (e.g., optical density). Plot the signals on the y-axis and the known concentrations on the x-axis to create the curve,

What is the purpose of a standard curve (using ELISA as an example)

standard curve is used in ELISA to determine the concentration of an unknown sample. After measuring the sample's signal, find the corresponding concentration on the curve by matching the signal to the plotted data. It defines the relationship between the signal (e.g., optical density) and known concentrations of the target analyte. By providing this baseline, the standard curve ensures that unknown sample measurements can be accurately converted into concentrations.

Antigen

Any substance that causes the body to make an immune response against that substance. Antigens include toxins, chemicals, bacteria, viruses, or other substances that come from outside the body.

Immune system

a complex network of organs, cells and proteins that defends the body against infection, whilst protecting the body's own cells

B lymphocytes

white blood cells that produce antibodies to help the body fight off bacteria, viruses, and toxins: 

Explain why antibodies allow scientists to target and identify specific disease agents.

they possess an incredibly high degree of specificity, meaning each antibody only binds to a unique molecular feature (called an antigen) on a specific pathogen, essentially acting like a "molecular key" that only fits one lock, thus allowing for precise identification of the disease agent involved

Why is the secondary antibody used in an ELISA test conjugated with an enzyme? What happens when this enzyme meets up with its substrate?

it catalyzes a chemical reaction, usually resulting in a color change that indicates the presence of the target antigen in the sample, allowing for quantification of the antigen level.

What are the steps of ELISA?

1. Step 1: Prep the Base (Coating)

   • Imagine applying primer to your face. In ELISA, the plate gets "primed" with the antigen or antibody to stick to the surface.

   • If everything is sticking, we’re off to a good start!

2. Step 2: Block That Plate (Blocking)

   • Like putting on moisturizer to stop makeup from caking, we add a blocking solution to prevent random stuff from sticking to the plate. This ensures only the important interactions happen.

3. Step 3: Add the Main Ingredient (Primary Antibody)

   • Time for the serum! The primary antibody is applied, and it binds specifically to the target antigen. This is the key step for the glow-up.

4. Step 4: Boost It (Secondary Antibody)

   • Now for the glow booster! Add the secondary antibody, which has the enzyme attached. It’s like adding highlighter to make everything pop.

5. Step 5: Reveal the Glow (Substrate)

   • Finally, apply the color-changing substrate. The enzyme reacts with the substrate, and the plate "glows" (changes color) where the target is present.

6. Step 6: Snap the Pic (Detection)

   • Measure the glow (optical density) in a plate reader. The brighter the glow, the more target you have in the sample.

What could cause ELISA results to show false negatives or false positives?

• Improper Blocking:

  • Without proper blocking, non-specific proteins stick to the plate, causing false positives.

• Insufficient Washing:

  • Leftover unbound antibodies or reagents can cause non-specific signals, leading to inaccurate results.

• Contaminated Reagents:

  • Contaminated antibodies, substrates, or buffers can introduce unwanted reactions.

• Expired Reagents:

  • Old or improperly stored reagents may lose activity, giving weak or inconsistent signals.

• Incorrect Dilutions:

  • Over- or under-diluting antibodies or samples can skew results, causing weak signals or false positives.

• Cross-Reactivity:

  • Antibodies binding to unintended targets in the sample can create misleading results.

• Improper Plate Coating:

  • If the antigen or antibody isn’t evenly coated, the signal may be inconsistent.

• Substrate Issues:

  • Using the wrong substrate or exposing it to light/heat before use can affect color development.

• Instrument Errors:

  • A miscalibrated plate reader can produce unreliable optical density readings.

Disease samples from two patients were collected and subjected to serial dilutions before running an ELISA. What does it mean if the disease can be detected in samples from one person only at a dilution of 1/5, but the disease can be detected in the other patient at a dilution of 1/5 and 1/100?

If the disease can only be detected in the first patient at a 1/5 dilution, but in the second patient at both 1/5 and 1/100 dilutions, it suggests the first patient has a lower concentration of the disease-related marker. The second patient’s sample contains a higher concentration of the marker, as it remains detectable even at a more diluted concentration (1/100). This difference indicates that the second patient has more of the disease-related marker in their sample.

Describe a situation that illustrates why it is a good idea to complete the ELISA in triplicate.

Running ELISA in triplicate ensures reliability by averaging results, reducing the effect of any random errors or anomalies in one of the samples. For example, if one of the wells shows an unusually high or low signal due to technical issues, triplicate testing helps confirm the true result and improves accuracy.

Why do you think college students living in dorms are populations that often see meningitis outbreaks?

College dorms are high-density living environments where close contact among students increases the risk of spreading meningitis-causing bacteria or viruses. Shared spaces like bathrooms and kitchens also contribute to the transmission, making outbreaks more common in these settings.

How did ELISA data allow you to track the path of infection at the college?

ELISA data provides measurable signals for the presence of specific antigens or antibodies in student samples. By tracking changes in antigen levels over time, researchers can identify when individuals were first exposed to the infection and how the disease spread through the population.

: Discuss the limitations of using antigen concentration to deduce the path of infection. Be sure to refer to the workings of the human immune system.

While antigen concentration can indicate exposure to a pathogen, it may not reveal the timing or full extent of infection. The immune system produces antibodies in response to antigens, and antigen levels can decline as the body clears the infection. Thus, antigen concentration alone may not give a complete picture of the infection’s path.

The ELISA test can also be used to detect antibodies that are produced in response to a specific antigen. Using information about how you completed this ELISA experiment, outline a procedure to test for antibodies in the blood.

To test for antibodies, coat the ELISA plate with a known antigen. Then, add the patient’s blood sample. If antibodies are present, they will bind to the antigen. Add a secondary antibody linked to an enzyme that binds to the primary antibody. Add a substrate, and if the antibodies are present, the plate will change color.

Explain why in sudden outbreaks, it may be better to test for disease antigens than for antibodies.

In sudden outbreaks, testing for antigens is preferable because antigens appear early in infection, while antibodies are produced later as the immune system responds. Testing for antigens can provide quicker detection of active infection, helping to identify individuals who are currently contagious.

Home pregnancy tests use ELISA technology. When a woman is pregnant, her body produces a hormone called human chorionic gonadotropin (hCG). Explain how antibodies can be used to detect this hormone and are linked to the color change a woman may see on a positive test.

In a home pregnancy test, the test strip is coated with antibodies specific to hCG. If hCG is present in the urine, it binds to these antibodies. This binding triggers a color change due to an enzyme-linked reaction, indicating a positive result.

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Hans Christian Gram

In 1884, while examining lung tissue from patients who had died of pneumonia, a microbiologist named Hans Christian Gram (Figure 1) discovered that certain stains were preferentially taken up and retained by bacterial cells. But Gram did not use a counterstain in his procedure.

It was a few years later, when the German pathologist Carl Weigert (1845–1904) from Frankfurt, added a final step of staining cells with safranin. Gram himself never used the red counterstaining to visualize the gram negative bacteria. Today, the Gram stain technique remains one of the first steps in classifying or identifying bacteria.^1,2

Antibiotics

medicines that fight bacterial infections in people and animals. They work by killing the bacteria or by making it hard for the bacteria to grow and multiply. Antibiotics can be taken in different ways: Orally (by mouth).

Nucleoid

The region where the bacterial DNA is located, containing the genetic material that controls the cell's activities and reproduction.

Plasmid

• Small, circular DNA molecules separate from the chromosomal DNA, often carrying genes that can provide advantages, such as antibiotic resistance.

Ribosomes

Structures where protein synthesis occurs, translating genetic information into proteins needed for the cell’s functions.

Cell wall

rovides structural support and protection, maintaining the shape of the cell and preventing it from bursting due to osmotic pressure.

Plasma membrane (cell membrane)

A lipid bilayer that controls the movement of substances into and out of the cell, maintaining the internal environment.

Capsule

A sticky layer that surrounds some bacteria, offering protection from the immune system and aiding in attachment to surfaces.

Flagellum:

A long, whip-like structure that enables bacteria to move (motility), often helping them move toward nutrients or away from harmful substances.

Pili:

short, hair-like projections on the cell surface that assist in attachment to surfaces and play a role in bacterial conjugation (gene transfer between cells).

Endotoxins

Toxic molecules found in the outer membrane of some bacteria (especially Gram-negative), which can trigger immune responses and cause inflammation when released.

What cellular components do some bacterial cells have that make them powerful pathogens?

• Capsule: A protective outer layer that helps bacteria evade the host’s immune system by preventing phagocytosis (engulfment by immune cells).

• Endotoxins: Toxic molecules, usually found in the outer membrane of Gram-negative bacteria, that can trigger severe immune responses, leading to inflammation, fever, and potentially shock.

• Exotoxins: Proteins secreted by bacteria that can damage tissues or disrupt normal cellular processes, often causing illness (e.g., botulinum toxin, cholera toxin).

• Pili (Fimbriae): Hair-like structures that help bacteria adhere to host tissues, which is crucial for infection establishment.

• Flagella: Tail-like structures that allow bacteria to move toward favorable environments (chemotaxis) or away from harmful substances, aiding in their spread within the host.

• Plasmids: Small DNA molecules that may carry genes for antibiotic resistance, enabling bacteria to survive treatments and persist in the host.

• Protein Secretion Systems: Complex mechanisms (e.g., Type III secretion system) that allow bacteria to inject virulence factors directly into host cells, disrupting their function or immune response.

• Biofilms: Groups of bacteria that stick to surfaces and form protective layers, often making them more resistant to antibiotics and immune defenses.

How do B-Lactams work?

These antibiotics are like the ultimate party crashers—they mess with the bacteria’s cell wall construction, stopping them from building their protective barrier. Without a strong wall, the bacteria burst open and can’t survive. Total microscopic meltdown.

How do Tetracyclines work?

Tetracyclines are like a squad of rebels—they bind to bacterial ribosomes (the protein factories) and stop them from making the proteins they need to grow. Without these essential proteins, the bacteria can't function, so they just can't even.

How do Fluoroquinolones work?

Fluoroquinolones are the ultimate hackers. They target the DNA-replicating machinery, especially enzymes like DNA gyrase. By blocking DNA from replicating, the bacteria literally can't copy themselves, and they’re left in a total panic—no new bacteria, no survival. Bye, bacteria!

How do sulfonamides work?

Sulfa antibiotics are like the sneaky sabotage artists—they block bacteria from making folic acid, a key nutrient they need for DNA and protein production. Without folic acid, the bacteria can't make the essentials for growth. It's like cutting off the supply chain for their survival.

Each of these antibiotics has its own way of taking down bacteria, from stopping their growth to breaking down their defenses. It's like a full-on bacterial smackdown!

Is Neisseria menigitidis gram-negative or gram-positive?

Gram negative

Why are penicillins often more effective against Gram-positive bacteria than Gram-negative bacteria?

Penicillins are more effective against Gram-positive bacteria because these bacteria have a thick peptidoglycan layer in their cell walls that is easily targeted by penicillin. Penicillin works by inhibiting the enzymes that help build and maintain the bacterial cell wall, which leads to cell lysis. Gram-negative bacteria, on the other hand, have a much thinner peptidoglycan layer and an additional outer membrane that acts as a barrier, making it harder for penicillin to penetrate and reach the cell wall.

. Why is it important to understand the structure of a bacterial cell when developing an antibiotic?

Understanding the structure of a bacterial cell is crucial because antibiotics target specific components of the bacterial cell. For example, penicillins target the cell wall, while others may target the ribosomes or DNA replication machinery. If we don’t understand how the cell is built and operates, it would be difficult to design antibiotics that effectively disrupt bacterial functions without harming human cells, which have different structures.

. How do antibiotics work without harming the surrounding human cells?

Antibiotics are designed to target features unique to bacteria, such as their cell walls, bacterial ribosomes, or specific enzymes. Human cells don’t have cell walls, and their ribosomes are structurally different from those of bacteria, allowing antibiotics to specifically target bacteria without damaging human cells. This selectivity is what makes antibiotics effective against bacteria without harming the human host.

What class of antibiotics would you prescribe for Sue? Explain your answer.

Broad-spectrum antibiotics or antibiotics meant to target gram-negative bacteria because prescribing her with bacteria that isn’t effective against gram-negative bacteria might lead to antibiotic resistance.

Why are antibiotics NOT effective against viruses?

Antibiotics are designed to target specific features of bacteria, such as cell walls or ribosomes, which viruses do not have. Viruses are made up of genetic material (DNA or RNA) and a protein coat, and they don’t rely on structures that antibiotics can target. Instead, viruses invade host cells and use the host's cellular machinery to reproduce, so antibiotics cannot interfere with viral replication. Antiviral medications, which target the viral life cycle, are required for treating viral infections.

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Why is it dangerous to listen to music at excessively loud volumes for extensive periods? 

• Damage to Hair Cells in the Inner Ear: The sound waves from loud music travel through the ear and can damage the tiny hair cells in the cochlea (inner ear) that are responsible for transmitting sound signals to the brain. These hair cells do not regenerate, so once they’re damaged, the loss of hearing is permanent.

• Noise-Induced Hearing Loss (NIHL): Prolonged exposure to loud sounds (typically above 85 decibels) can lead to a condition called noise-induced hearing loss. This condition worsens over time with continued exposure to high volumes.

• Tinnitus: Listening to loud music can also lead to tinnitus, a ringing or buzzing sound in the ears that can become persistent and debilitating.

• Distortion of Sound Perception: Over time, exposure to loud music can distort how you perceive sound, making it harder to hear certain frequencies or distinguish between different sounds.

outer ear

The outer visible portion of the ear that collects and directs sound waves toward the tympanic membrane by way of a canal which extends inward through the temporal bone

inner ear

The essential part of the vertebrate organ of hearing and equilibrium that includes the vestibule, the semicircular canals, and the cochlea.

middle ear

The intermediate portion of the ear containing a chain of three ossicles that extends from the tympanic membrane to the oval window and transmits vibrations to the inner ear.

Pinna

The cute outer part of your ear that you can see! It’s like a little funnel that catches sound waves and sends them into your ear. It helps you hear from all around you.

Auditory Canal:

Think of this as the ear’s “tunnel.” It's the path that directs sound from the pinna to your eardrum. No sound escapes—everything goes through here!

Eustachian Tube

This one’s like your ear’s secret little drain. It connects your middle ear to the back of your throat and helps balance pressure between your middle ear and the outside world. It's especially helpful when you’re on a plane or swimming!

Ossicles (Malleus, Incus, Stapes):

These are the three tiny bones in your middle ear that work together to amplify sound. They’re like the VIP squad of sound transmission:

• Malleus (hammer): The first to jump into action, vibrating from the eardrum.

• Incus (anvil): The middle member, passing vibrations along.

• Stapes (stirrup): The final bone, passing vibrations to the inner ear.

Tympanic Membrane (Eardrum):

This is the ultimate sound receiver—like a thin, super-sensitive drum that vibrates when sound hits it, sending the vibrations to the ossicles. It’s your ear’s very own concert hall.

Cochlea:

The cochlea is like your ear’s magical sound processor. It’s a spiral-shaped organ in your inner ear that turns vibrations into electrical signals that your brain can understand. It's kind of like your own internal concert, transforming sound into a language your brain gets.

Sensory Hair Cells:

These tiny hairs inside the cochlea are the real stars. They detect vibrations and convert them into nerve signals. The more hairs that move, the louder the sound. Too much noise? Some of these hairs might not come back, so it’s important to protect them!

Cochlear Nerve:

After the sensory hair cells send their signals, the cochlear nerve is like the messenger. It carries all those signals from the cochlea to your brain so you can hear everything clearly.

Oval Window:

This is the entrance to the cochlea, the place where the stapes bone sends vibrations in. It’s like the door to the inner ear that lets sound waves into the cochlea for processing.

Vestibule:

The vestibule helps with balance! It’s located next to the cochlea and works with the semicircular canals to help you stay steady and balanced. No falling over, girlypop!

Vestibular Nerve:

This nerve is your balance buddy. It sends signals from the vestibule and semicircular canals to your brain so you can stay upright and in control, whether you're standing still or spinning around.

What are the steps of hearing?

• Sound Waves Enter the Ear:
  The pinna (outer ear) acts like a sound-catching funnel, collecting sound waves from the environment. These sound waves then travel through the auditory canal to reach the eardrum (tympanic membrane).

• Vibrations are Transferred:
  When the sound waves hit the eardrum, it vibrates. These vibrations are passed to the three tiny bones in the middle ear called the ossicles (malleus, incus, and stapes). The ossicles amplify the vibrations and pass them along to the next part.

• Vibrations Enter the Inner Ear:
  The stapes (the last ossicle) pushes against the oval window, a small membrane at the entrance of the cochlea. This action creates waves in the fluid inside the cochlea, which causes the sensory hair cells in the cochlea to move.

• Signal is Sent to the Brain:
  The sensory hair cells in the cochlea turn the movement into electrical signals. These signals travel through the cochlear nerve to the brain, where they’re interpreted as sound, allowing you to hear and understand what’s going on around you.

Sound

Mechanical energy that is transmitted by longitudinal pressure waves in a medium (such as water or air).

Sound intensity is measured

decibels (dB).-this unit measures amplitude of a sound wave

Amplitude controls

the loudness (how intense the sound is).

Frequency controls the

pitch (how high or low the sound is).

What measures frequency?

Hertz (Hz)

What is a serial dilution?

is a process used in science to dilute a substance (like a solution or sample) step by step, typically in half or by a certain factor, to get progressively weaker concentrations. Here's how it works, girlypop style:

How do I solve serial dilution word problems?

"Step By Step, Take It Slow"

• Step: Think about the starting concentration. This is where you begin.

• By: This is for the dilution factor. (e.g., 1:2 means you're diluting by half each time).

• Slow: This reminds you to do it step-by-step. Don't rush! Each dilution is a fraction of the previous step. For example, if you're doing a 1:10 dilution, you’re multiplying the concentration by 1/10 each time.

How do I solve serial dilution word problems if I’m not given a dilution factor?

"Mix It Right, Divide It Tight!" 🎉✨

"Mix It Right" means figuring out how much of the stock solution is being mixed with the solvent.
"Divide It Tight" means dividing the amount of stock solution by the total volume (stock solution + solvent) to find the dilution factor.