Oral Revalida Flashcards
Biological agent
- any organism, virus, or toxin
- has the potential to cause
harm, disease, or some form of biologically mediated damage. - infectious pathogens like bacteria, viruses, fungi, or toxins produced by living organisms.
Biological material
- any substance derived from living organisms
- cells, tissues, proteins, enzymes, DNA, RNA, or any other biological component.
Standard Precautions
“Treat ALL human blood samples and other body fluids as infectious”
- Healthcare professionals and laboratory workers are trained to
handle these samples with utmost care, using appropriate personal protective equipment (PPE) .
- Proper handling, labeling,
transportation, and disposal procedures are followed strictly to minimize accidental
exposure or contamination.
- fundamental part of infection control practices
Biosecurity
Deals with preventing intentional harm or misuse of biological agents
Biosafety
Preventing accidental exposure or harm to
individuals and the environment when handling biological agents
Similarities of Biosecurity and Biosafety
Crucial in managing biological materials responsibly and safely in various settings.
Serves as the storage of genetic information
DNA
intermediary role in
translating that information into proteins.
RNA
What are the differences between DNA and RNA?
- Both DNA and RNA are crucial in the central dogma of molecular biology, where DNA
serves as the storage of genetic information, and RNA plays an intermediary role in
translating that information into proteins. - DNA consists of a double-stranded helical structure. While RNA is usually single-stranded.
Two types of nitrogenous bases that are essential
components of nucleotides, the building blocks of DNA and RNA.
Pyrimidines and purines
Pyrimidines
- smaller, single-ring nitrogenous bases
- cytosine (C), thymine (T, found in DNA), and uracil (U, found in RNA)
- consist of a six-membered ring made up of four carbon atoms and two nitrogen atoms.
Purines
- larger, double-ring nitrogenous bases
- They consist of a six-membered ring fused (attached) to a five-membered ring.
- two purine bases: adenine
(A) and guanine (G).
DNA replication
- Process by which a cell makes an exact copy of its DNA before
cell division. - It occurs during the S (synthesis) phase of the cell cycle
Steps of DNA replication process
- Unwinding
- Initiation
- Primer Binding
- Elongation
- Proofreading and Correction
- Ligase Activity
- Termination
Unwinding (DNA Replication process)
- DNA double helix is unwound by enzymes called helicases.
- These enzymes break the hydrogen bonds between the complementary base pairs, separating the two DNA strands and creating a replication fork.
Initiation (DNA Replication process)
- Enzymes known as DNA polymerases recognize specific sequences called origins of replication along the DNA strands.
- These polymerases initiate
the replication process by binding to the DNA at these sites.
Primer Binding (DNA Replication process)
- RNA primers are synthesized by another enzyme called primase.
- it provides a starting point for DNA polymerases to begin adding new nucleotides.
Elongation (DNA Replication process)
- DNA polymerases start adding complementary nucleotides to the
exposed DNA strands. They can only add nucleotides in the 5’ to 3’ direction, so one strand (the leading strand) is synthesized continuously in the direction of the replication fork. - The other strand (the lagging strand) is synthesized discontinuously in short segments called Okazaki fragments, as it must be synthesized in the opposite direction of the replication fork.
Proofreading and Correction (DNA Replication process)
- DNA polymerase is utilized
- It adds nucleotides, they can recognize and correct mistakes in base pairing,
ensuring accuracy in the newly synthesized DNA.
Ligase Activity (DNA Replication process)
Once the new nucleotides are added, DNA ligase seals the nicks
or gaps between the Okazaki fragments, joining them together into a continuous
strand.
Termination (DNA Replication process)
- The replication continues bidirectionally along the DNA until the
entire DNA molecule is replicated. - The process ends when the replication forks
meet, and the entire DNA molecule has been duplicated.
After DNA replication, what will happen?
- Two identical DNA molecules are formed, each containing one original
strand and one newly synthesized strand. - This semiconservative replication ensures
genetic continuity and fidelity in daughter cells during cell division.
Protein synthesis
Process by which cells generate new proteins based on the
genetic information encoded in DNA.
Main stages of Protein Synthesis
- Transcription
- Translation
Stages of Transcription
- Initiation
- Elongation
- Termination
Initiation (Transcription)
- The process begins in the nucleus of eukaryotic cells or the
cytoplasm of prokaryotic cells. - RNA polymerase, along with various
transcription factors, binds to a specific region of DNA called the
promoter. - Promoter marks the start of transcription.
Elongation (Transcription)
- RNA polymerase moves along the DNA strand, unwinding the double helix and synthesizing a complementary RNA strand.
- The enzyme
adds complementary RNA nucleotides (adenine, cytosine, guanine, and
uracil) based on the DNA template. - RNA nucleotides pair with their
complementary bases on the DNA template
What will happen after the Transcription process?
The newly formed RNA molecule, known as messenger RNA (mRNA), carries the genetic information from the DNA in the nucleus to the cytoplasm, where protein synthesis continues.
initiation (Translation)
- mRNA binds to a ribosome, and the process of
translation begins. - Ribosomes serve as the site for protein synthesis.
- Initiation factors and the start codon (AUG) on the mRNA signal the
recruitment of the first transfer RNA (tRNA) molecule carrying the amino
Elongation (Translation)
- Ribosome moves along the mRNA
strand in a 5’ to 3’ direction, reading the genetic code in sets of three
nucleotides called codons. - Each codon corresponds to a specific amino acid carried by a tRNA molecule. - The ribosome facilitates the binding of
the appropriate tRNA carrying the correct amino acid to the mRNA codon
via complementary base pairing.
Peptide Bond Formation (Translation)
- As each tRNA brings its amino acid in sequence, a peptide bond forms between adjacent amino acids, catalyzed by the ribosome.
- Forms a growing polypeptide chain.
Termination (Translation)
- Translation continues until a stop codon (UAA, UAG, or UGA)
is encountered on the mRNA which signal the end of protein synthesis. - Release factors bind to the
ribosome, causing the completed polypeptide chain to be released
DNA extraction
- Process of isolating DNA from cells or tissues in a biological
sample. - Allows scientists to study and analyze the genetic material.
Sample Collection
Biological samples, such as blood, saliva, tissues, plants, bacteria, or cells,
are collected and stored properly to preserve the DNA.
Cell lysis
- The first step involves breaking open the cells to release the DNA.
- This is achieved through a process called lysis, where cells are disrupted using various methods.
Methods for Cell Lysis
Mechanical methods like grinding, crushing, or homogenization, and
chemical methods using detergents or enzymes can be employed to break
down cell membranes and release the DNA.
Removal of Proteins and RNA:
Enzymes like proteases are used to degrade proteins, and RNases break down RNA, leaving only DNA in the solution.
DNA precipitation
- To separate the DNA from other cellular components, a precipitation step
is performed. - This involves adding a salt solution (such as sodium chloride) and alcohol (like ethanol or isopropanol) to the DNA-containing solution. This causes the DNA to clump together and precipitate out of the solution.
DNA Purification
- The precipitated DNA is then collected by centrifugation, forming a pellet
at the bottom of the tube. - The supernatant (liquid above the pellet) is discarded,
- The DNA pellet is washed with alcohol to remove any
remaining impurities or salts.
Resuspension
The purified DNA pellet is then resuspended or dissolved in a suitable
buffer or solvent, such as Tris-EDTA (TE) buffer or distilled water, to obtain
a concentrated DNA solution
DNA EXTRACTION
- Sample Collection
- Cell Lysis
- Removal of Proteins and RNA
- DNA Precipitation
- DNA Purification
- Resuspension
RNA isolation
- Process of extracting and purifying RNA molecules from cells, tissues, or biological samples.
- Allowing scientists to study gene expression, RNA structure, and function.
RNA ISOLATION STEPS
- Sample Collection and Preservation
- Cell Lysis and RNA Stabilization
- Separation of RNA from DNA and Proteins
- RNA Extraction and Purification
- RNA Precipitation and Washing
- RNA Resuspension
Sample Collection and Preservation (RNA isolation)
- Collect the biological sample (cells, tissues, blood, etc.) and preserve it
immediately to prevent RNA degradation. - RNA is highly susceptible to
degradation by RNases (ribonucleases) present in the environment.
Cell Lysis and RNA Stabilization (RNA Isolation)
- Break open the cells to release the RNA through mechanical disruption (homogenization, grinding), chemical lysis (detergents), or enzymatic lysis.
- To prevent RNA degradation, RNA stabilizing agents like RNase inhibitors
or chaotropic salts are often added to the lysis buffer.
Separation of RNA from DNA and Proteins (RNA Isolation)
- RNA needs to be separated from other cellular components like proteins and DNA.
- Enzymes like DNase (to degrade DNA) and proteases (to degrade
proteins) are used to remove DNA and proteins from the RNA solution.
RNA Extraction and Purification (RNA isolation)
- RNA extraction typically involves the addition of an organic solvent (e.g.,
phenol-chloroform) and subsequent centrifugation to separate the RNA
from other components. - After extraction, the RNA-containing aqueous phase is carefully collected
and transferred to a new tube.
RNA Precipitation and Washing (RNA isolation)
The RNA is precipitated by adding alcohol (such as isopropanol or
ethanol) which RNA molecules forms visible pellet, as it remove residual salts and contaminants.
RNA Resuspension (RNA isolation)
Purified RNA pellet is then dissolved or resuspended in a suitable
buffer or solvent, such as RNase-free water or RNase-free buffers, to
obtain a concentrated RNA solution.
DNA extraction
- Isolate genomic DNA from cells or tissues.
- Genomic DNA contains the genetic information necessary for an organism’s hereditary
traits. - Isolated DNA is used in various applications such as PCR, DNA sequencing, cloning, genotyping, and genetic analysis.
RNA isolation
- Aims to extract RNA molecules.
- Play roles in gene expression,
protein synthesis, and regulation within cells. - Isolated RNA is used for gene expression studies, mRNA quantification, microarray analysis, RNA sequencing (RNA-seq), and other molecular biology techniques focused on understanding gene regulation and expression.
What is Hybridization?
- a fundamental principle used in molecular biology techniques to study,
detect, and manipulate nucleic acids by exploiting the specificity of base pairing
between complementary sequences. - enables the detection, characterization, and analysis of specific DNA or RNA sequences critical in understanding genetics, gene
expression, and various biological processes.
What is Southern Blot?
- powerful tool in molecular biology for identifying
and analyzing specific DNA sequences within a sample. - widely used in
genetic research, DNA fingerprinting, gene mapping, and various applications aimed at
understanding DNA structure and function.
What is Northern Blot?
- For studying gene expression, identifying and quantifying specific RNA transcripts, examining RNA sizes, splice variants, and exploring RNA processing and regulation in various biological systems
- RT-PCR, RNA sequencing,
and microarrays have become more commonly used for RNA analysis due to their higher sensitivity and throughput capabilities.
What is Western Blot?
- commonly used in molecular biology and biomedical
research to detect and analyze proteins, study protein expression, verify protein size, confirm protein identity, investigate post-translational modifications, and assess protein-protein interactions. - It’s a versatile tool that provides valuable information
about protein expression levels and characteristics within biological samples.
What is Dot Blot?
- Relatively quick and simple technique used for screening purposes,
detecting the presence of specific proteins or nucleic acids in a sample without the need for extensive sample preparation or gel separation. - it lacks the quantitative
accuracy of Western blotting or other more sophisticated methods, dot blotting is valuable for rapid screening and qualitative analysis.
Detects specific DNA sequences in a sample.
Southern Blot
Detects and analyzes specific RNA molecules in a sample.
Northern Blot
Detects and analyzes specific proteins in a sample.
Western Blot
Detects specific nucleic acids (DNA or RNA) or proteins, providing
qualitative information about the presence or absence of target molecules.
Dot Blot
What are the similarities between Southern Northern Blot, Western Blot, and Dot Blot?
All these techniques involve the transfer or direct spotting of samples onto a solid
support membrane (typically nitrocellulose or PVDF membrane), allowing target
molecules to be immobilized for subsequent analysis.
Fundamental guidelines and procedures followed in laboratory settings to ensure safety, accuracy, and efficiency in conducting experiments
and handling various substances.
Basic laboratory practices
Basic laboratory practices
Personal Protective Equipment (PPE)
Lab Hygiene
Handling Chemicals
Biological Safety
Equipment Use and Maintenance
Waste Management
Emergency Preparedness
Documentation and Record-Keeping
Training and Education
Communication and Collaboration
Wear appropriate PPE such as lab coats, gloves, safety goggles, and
closed-toe shoes to protect against chemical spills, splashes, or biological
hazards.
Personal Protective Equipment (PPE)
Maintain cleanliness in the lab by cleaning work surfaces before and after
use. Regularly decontaminate equipment and dispose of waste properly.
Lab Hygiene
Understand the properties and hazards of chemicals used in experiments.
Follow proper storage, handling, and disposal procedures. Label all
containers clearly.
Handling Chemicals
Adhere to protocols for handling biological materials. Use proper
containment, sterilization, and disposal methods to prevent contamination
and exposure
Biological Safety
Calibrate and maintain laboratory equipment regularly to ensure accuracy
and reliability of results. Follow instructions for safe use of machinery and instruments.
Equipment Use and Maintenance
Dispose of laboratory waste, including chemicals, biological materials, and
sharps, according to regulations and established guidelines.
Waste Management
Know the location and proper use of emergency equipment such as fire
extinguishers, eyewash stations, and emergency showers. Be familiar with
emergency procedures and evacuation routes.
Emergency Preparedness
Maintain accurate records of experiments, observations, and results in laboratory notebooks or electronic records. Properly label samples and
data.
Documentation and Record-Keeping
Ensure that laboratory personnel are adequately trained in safety
protocols, handling techniques, and emergency procedures. Regularly
update training on new procedures or hazards.
Training and Education
Foster open communication among lab members to share information,
address safety concerns, and collaborate effectively on experiments.
Communication and Collaboration
What is the importance of following the laboratory practices?
Following these practices helps to create a safe, controlled, and productive laboratory environment, reducing risks and ensuring the reliability of experimental outcomes.
Forward pipetting
- Commonly used for accurate measurement and transfer of liquids.
- Liquid is drawn into the pipette tip by aspirating the desired
volume into the tip and then dispensing it into another container
Reverse pipetting
- Useful when handling viscous, volatile, or foamy liquids.
- After aspirating the liquid into
the pipette tip, the plunger is pushed past the first stop, expelling the entire volume back into the source container.
General Procedure on Preparation of Reagents
- Gather Equipment and Materials
Read the Protocol or Recipe - Read the Protocol or Recipe
- Calculate Quantities
- Prepare the Solution
- Adjust pH or Concentration (if necessary)
- Check and Verify
- Label and Store
- Document and Record
- Clean-Up
What is Spectrophotometry?
- It’s used to determine the concentration of substances like DNA, proteins, enzymes, drugs, and
various compounds in solutions. - It measures the abosrption of light to determine the concentration of a substance.
How does Spectrophotometry works?
- A sample solution is placed into a cuvette and inserted into the
spectrophotometer. - The spectrophotometer emits light of specific wavelengths through the sample.
- The detector measures the intensity of light that passes through the sample and
compares it to the intensity of the initial light source. - The spectrophotometer calculates the absorbance or transmittance of the
sample at each wavelength. - The absorbance is then used to determine the concentration of the substance in
the solution using Beer-Lambert’s law, which relates absorbance to
concentration.
Polymerase Chain Reaction (PCR)
-a powerful molecular biology technique used to amplify a specific segment of DNA (or RNA) to generate millions to billions of copies.
- This method was developed by Kary Mullis in the 1980s and has since become a fundamental tool in various scientific fields, diagnostics, forensics, and genetic
research
PCR Steps
Denaturation
Annealing
Elongation
Denaturation
- The double-stranded DNA template is heated to a high temperature (typically around 94-98°C), causing the DNA strands to separate and denature into two single strands.
Annealing
- The reaction temperature is lowered (usually to around 50-65°C),
- Allowing short DNA primers—complementary to sequences flanking the target region—to bind (anneal) to their specific locations on the single-stranded DNA template.
Elongation
- The temperature is raised to the optimal working temperature of the
DNA polymerase (usually around 72°C). - Adding nucleotides to the 3’ end of each primer, synthesizing new
DNA strands complementary to the template.
Taq DNA Polymerase
From the Thermus aquaticus found in hot springs. It is heat-stable and active at high temperatures, making it ideal for PCR.
RT-PCR
“Reverse Transcription Polymerase Chain Reaction”
- Used to amplify and detect RNA molecules, specifically messenger
RNA (mRNA), by converting RNA into complementary DNA (cDNA) through a process called reverse transcription, followed by PCR amplification of the cDNA.
RT-PCR has various applications in molecular biology, diagnostics, and research, including:
- Gene expression analysis
- Viral load quantification
- Disease diagnosis
- Studying RNA viruses
Gene expression analysis
Quantifying gene expression levels by measuring mRNA levels.
Viral load quantification
Detecting and quantifying viral RNA for viral load assessment, such as in HIV or SARS-CoV-2 detection.
Disease diagnosis
Identifying genetic disorders or diseases associated with specific RNA transcripts.
Studying RNA viruses
Analyzing RNA viruses and their replication processes.
What is Multiplex PCR?
- It is an advanced variant of the traditional PCR technique that allows for the simultaneous amplification of multiple target DNA or RNA sequences in a single reaction.
- Multiplex PCR uses multiple pairs of primers, each designed to amplify different target sequences.
Where is Multi PCR used?
It is widely used in various fields, including pathogen detection, genotyping, gene
expression analysis, forensic analysis, and mutation screening, where multiple targets need to be analyzed simultaneously.
Quantitative Polymerase Chain Reaction (qPCR)
- Also known as real-time PCR, is a
molecular biology technique used to amplify and simultaneously quantify a specific DNA or RNA target sequence during the PCR process.
Application of qPCR
● Gene expression analysis
● Quantification of DNA or RNA viruses
● Pathogen detection (bacteria, viruses, fungi)
● Environmental monitoring
● Genetic testing and disease diagnosis
Nested PCR (Polymerase Chain Reaction)
- used for increased specificity in amplifying a target DNA sequence,
especially from samples containing limited or degraded DNA - uses two sets of primers to amplify the
target DNA sequence.
Nested PCR application
● Amplification of specific genes from complex samples (e.g., forensic or ancient
DNA analysis)
● Detection of pathogens in clinical samples with low pathogen levels
● Amplification of low-abundance or rare target sequences
Digital PCR (dPCR)
“Digital Polymerase Chain Reaction”
- used for precise and absolute quantification of nucleic
acids (DNA or RNA) in a sample.
- dPCR partitions the sample into thousands of
individual micro-reactions, allowing for the absolute quantification of target nucleic acid
Application of dPCR
● Quantification of gene expression levels
● Detection and quantification of rare genetic mutations or variants
● Absolute quantification of viral load in clinical samples
● Copy number variation analysis
● Environmental and food testing
Nucleic Acid Sequence-Based Amplification (NASBA)
- used to amplify RNA sequences.
- requires thermal cycling between different temperatures.
- NASBA operates at a constant
temperature, making it an isothermal amplification method.
What is Strand Displacement Amplification?
-Used to amplify DNA sequences.
- Developed as an alternative to PCR, SDA operates at a constant temperature without the need for thermal cycling
Application of Strand Displacement Amplification
● Infectious disease diagnostics
● Pathogen detection, especially for bacterial and viral DNA
● Gene detection and analysis
● Environmental and food testing for DNA-based pathogens
What is Rolling Circle Amplification?
- an isothermal nucleic acid amplification technique primarily used to amplify circular DNA templates.
- It operates at a constant temperature, producing long single-stranded
DNA (ssDNA) or RNA molecules that form repetitive sequences through repeated rolling circle replication.
Application of Rolling Circle Amplification
● DNA and RNA detection assays
● Pathogen detection, especially for circular viral genomes
● Amplification of circularized DNA molecules in cloning or library preparation
● Aptamer generation and molecular probes
What is Recombinase Polymerase Amplification?
- An isothermal nucleic acid amplification technique that rapidly amplifies DNA or RNA targets at a constant, low temperature.
- It’s designed to replicate specific DNA or RNA sequences with high sensitivity and specificity, often used for diagnostic purposes and field applications due to its rapid and simple workflow.
Application of Recombinase Polymerase Amplification
● Molecular diagnostics, especially for infectious disease detection (viral, bacterial,
or parasitic)
● Environmental monitoring
● Food safety and pathogen detection
● Forensic analysis
What is Helicase-Dependent Amplification?
- Is an isothermal nucleic acid amplification technique used to amplify DNA targets at a constant temperature without the need for thermal cycling, making it suitable for rapid
and sensitive molecular diagnostics.
Application of Helicase-Dependent Amplification
● Molecular diagnostics for infectious diseases
● Pathogen detection (viruses, bacteria, parasites)
● Point-of-care testing
● Environmental monitoring
● Food safety and quality control
What is Polymerase Spiral Reaction?
- an isothermal nucleic acid amplification technique designed for the rapid and sensitive detection of DNA or RNA targets at a constant
temperature. - It operates without the need for thermal cycling equipment, making it
suitable for point-of-care diagnostics and field applications.
What is Gel Electrophoresis?
It is a versatile tool that allows scientists to separate and analyze biomolecules based on their distinct physical properties, aiding in numerous research, diagnostic, and forensic applications.
Separating DNA fragments (Gel Electrophoresis)
DNA fingerprinting, genetic analysis, or DNA sequencing.
Separating RNA fragments (Gel Electrophoresis)
Study gene expression or RNA size variants
Separating proteins (Gel Electrophoresis)
for analysis of protein size, purity, or identification.
Advantages of Gel Electrophoresis
- Allowing the separation and analysis of biomolecules based on size, charge, or properties.
- Biomolecule separation, versatility in applications like genetic
analysis, and quantification through distinct bands. - Accuracy in sizing and ease of use
are notable benefits.
Disadvantages of Gel Electrophoresi
- It may lack resolution for similar-sized molecules, betime-consuming, and pose challenges in fragile molecule handling.
- Protein separation can be technically demanding, and precision in quantitative measurements may be limited compared to advanced methods
Smiling Gel or Poor Band Resolution
● Cause: Uneven gel pouring, air bubbles, or improper gel polymerization.
● Solution: Ensure even gel pouring, eliminate air bubbles, and ensure
proper polymerization by allowing sufficient time for the gel to solidify
Fuzzy Bands or Smears on the Gel
● Cause: Overloaded samples, degraded DNA/RNA, or impurities in the
samples.
● Solution: Reduce sample concentration or volume, use fresh samples, and
ensure sample purity.
No Bands Visible on the Gel
● Cause: DNA/RNA or proteins did not migrate properly due to insufficient
voltage, wrong buffer, or improper handling.
● Solution: Check power supply and voltage settings, use the appropriate
buffer, and ensure correct handling during loading and running
Uneven Bands or Unequal Migration
● Cause: Uneven distribution of ions in the buffer, uneven loading of
samples, or irregular gel surface.
● Solution: Prepare buffer properly, load samples evenly, and ensure a level
gel surface before polymerization.
Bands Running Off the Gel or Smudging
● Cause: High voltage, overheating, or buffer depletion.
● Solution: Lower the voltage, use a cooling system if applicable, and ensure
the buffer level remains constant throughout the run.
High Background or Staining Issues
● Cause: Excess or incomplete staining, or contamination.
● Solution: Optimize staining protocols, use appropriate dye concentrations,
and ensure clean equipment and buffers to avoid contamination.
Buffer Leakage or Gel Falling Apart
● Cause: Improper sealing of the gel plates or inadequate gel strength.
● Solution: Ensure proper assembly of gel plates with appropriate seals, and
optimize gel concentration for the intended application.
No Marker Bands or Distorted Markers
● Cause: Inadequate loading or degradation of marker DNA/RNA or protein.
● Solution: Ensure proper loading of markers, use fresh and high-quality
markers, and handle them carefully to prevent degradation.
