Molecular Diagnostics Overview

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Questions and Answers

What is the primary focus of molecular diagnostics?

Detecting and measuring genetic material or proteins related to diseases (correct)

Analyzing demographic data in health care

Studying cellular structures and functions

Identifying environmental impacts on health

Which term describes the approach that tailors medical treatment to individual characteristics?

Generalized medicine

Epidemiological medicine

Preventive medicine

Personalized medicine (correct)

What is the role of companion diagnostics in molecular diagnostics?

To monitor long-term health outcomes for all patients

To provide general health advice

To enhance the effectiveness of a specific therapy for a patient (correct)

To diagnose diseases unrelated to genetic material

How can molecular diagnostics aid in family planning?

By identifying carriers of genetic mutations related to certain conditions Signup and view all the answers

What recent trend has been observed in the field of molecular diagnostics?

Increasing application in infectious diseases Signup and view all the answers

Which of the following best describes molecular diagnostics' application in blood banks?

To screen for infectious diseases in donated blood Signup and view all the answers

What is one of the benefits of molecular diagnostics in cancer treatment?

Helping to identify the likelihood of cancer recurrence Signup and view all the answers

Which statement about molecular diagnostics is correct?

They can identify multiple strains of viruses in a single test. Signup and view all the answers

What is the proper incubation temperature for lysing cells?

56°C Signup and view all the answers

What is the purpose of adding ethanol to the lysate?

To precipitate the DNA Signup and view all the answers

What should be done if residual solution is visible in the purification column after the second wash?

Re-spin the column for 1 minute at maximum speed. Signup and view all the answers

How is DNA concentration estimated?

By measuring absorbance at 260 nm and adjusting for turbidity. Signup and view all the answers

What is the expected A260/A280 ratio for pure dsDNA?

1.8 Signup and view all the answers

What is the maximum speed for centrifuging the GeneJET Genomic DNA Purification Column during the second wash?

12000 x g Signup and view all the answers

What should be done with the purification column after eluting the DNA?

Discard the purification column. Signup and view all the answers

How does reducing the volume of Elution Buffer affect the final quantity of eluted DNA?

It decreases the final quantity. Signup and view all the answers

What is the purpose of adding a concentrated salt solution during DNA extraction?

To make cellular debris clump together. Signup and view all the answers

Which method involves the denaturation of proteins to facilitate the separation of nucleic acids?

Phenol–chloroform extraction. Signup and view all the answers

What role does EDTA play in the lysis solution?

It chelates divalent cations and inactivates nucleases. Signup and view all the answers

Why is Proteinase K used during protein removal in DNA extraction?

It digests a wide range of native proteins. Signup and view all the answers

What is the primary purpose of using ice-cold ethanol or isopropanol in DNA purification?

To precipitate DNA by making it insoluble. Signup and view all the answers

What is the function of SDS in cell lysis?

It breaks apart membranes by interacting with lipids and proteins. Signup and view all the answers

How does minicolumn purification separate nucleic acids from other components?

By adsorption of nucleic acids to a solid phase. Signup and view all the answers

Which of the following is NOT typically part of a common lysis solution?

Sodium acetate Signup and view all the answers

What is the primary focus of the molecular diagnostic laboratory as outlined in the introduction?

Diagnostic testing and identification of molecular markers Signup and view all the answers

Which of the following is critical for ensuring lab safety in molecular biology?

All of the above Signup and view all the answers

What is the key purpose of preparing solutions in molecular diagnostics?

To dilute substances to specific concentrations Signup and view all the answers

What is the first step in the experimental protocol for genomic DNA preparation from blood?

Blood collection Signup and view all the answers

Why is the measurement of DNA concentration important in molecular diagnostics?

To ensure adequate amounts for subsequent testing Signup and view all the answers

Which of the following components is NOT typically included in the preparation of genomic DNA from blood?

Reagents for protein synthesis Signup and view all the answers

What is the significance of measuring the purity of DNA after extraction?

To ensure the absence of contaminants Signup and view all the answers

In which section of the molecular diagnostic lab notebook would you find the experimental protocol?

Preparation of Genomic DNA from Blood Signup and view all the answers

What is the primary purpose of the lysis step in silica-based DNA extraction?

To break down cells and access DNA Signup and view all the answers

Which component is added during the purification-bind step to enhance the binding of DNA to the silica membrane?

Ethanol Signup and view all the answers

What is the result of not removing all ethanol during the elution step?

The DNA cannot fully rehydrate Signup and view all the answers

During the wash step, what is the purpose of the two different washes with buffers?

To remove impurities and residual proteins and salts Signup and view all the answers

What is the recommended storage condition for blood samples prior to DNA extraction?

At room temperature or 4°C Signup and view all the answers

What role does chaotropic salt play in the lysis step?

It disrupts hydrogen bonds and hydrophobic interactions Signup and view all the answers

How can the elution of DNA be maximized during the procedure?

By allowing the elution buffer to sit in the membrane for a few minutes Signup and view all the answers

What is the purpose of including proteinase K in the DNA extraction protocol?

To digest proteins and facilitate lysis Signup and view all the answers

What is the purpose of the cell lysis solution in DNA extraction?

To break down cellular membranes and release DNA Signup and view all the answers

Which precaution is crucial to prevent contamination of isolated DNA?

Employing proper pipetting techniques Signup and view all the answers

How can heme contamination in isolated DNA be detected?

By performing a UV-Vis spectral analysis Signup and view all the answers

What method can be used to confirm the presence of DNA in a stored sample?

Conducting gel electrophoresis Signup and view all the answers

Is it feasible to prove that your DNA is actually in the sample?

Yes, through sequencing analysis Signup and view all the answers

How is DNA extraction significantly beneficial to forensic scientists?

It aids in the identification of individuals through DNA profiling Signup and view all the answers

What role does heme play as a contaminant in DNA isolation from blood?

It can interfere with PCR amplifications Signup and view all the answers

What is one major challenge faced in isolating DNA from blood samples?

Presence of cellular macromolecules Signup and view all the answers

What is a typical strategy to increase DNA purity during extraction?

Incorporating ethanol precipitation Signup and view all the answers

What is the primary goal during the DNA extraction process?

To ensure the DNA is free from any contaminants Signup and view all the answers

Which technique is often used to visualize DNA post-extraction?

Gel electrophoresis Signup and view all the answers

What can cause DNA degradation during the extraction process?

Prolonged exposure to light Signup and view all the answers

What consequence arises from improper pipetting during DNA extraction?

Risk of sample contamination Signup and view all the answers

What aspect of DNA does the purity assessment focus on during extraction?

Presence of contaminants Signup and view all the answers

Study Notes

Molecular Diagnostic Laboratory

Molecular biology is the study of biology at the molecular level, focusing on genetic material.
Molecular diagnostics is a dynamic field revolutionizing healthcare by detecting and measuring genetic material or proteins associated with specific diseases.
This allows for personalized medicine approaches, tailoring care to individual needs.
Molecular diagnostics discover biomarkers, leading to treatments designed around these markers.
This aids in accurate disease diagnosis, improving the delivery of therapies to the correct patients.
Molecular diagnostics are used in hospitals, reference labs, and blood banks.
They screen donated blood for infectious diseases and diagnose pathogens.
Increasing demand exists for point-of-care molecular diagnostic technologies.

Introduction

Molecular diagnostics are crucial in various fields including health, medicine, and forensics.
PCR-based technology, Fluorescence in situ hybridization (FISH), nanotechnology, biochips, peptide nucleic acid (PNA), and proteomic technology are used in diagnostics.
Electrochemical detection of DNA also is a molecular approach.

Safety Rules

Avoid eating, drinking, and smoking in the lab.
Wear protective gear (gloves, goggles, lab coat) when handling hazardous chemicals.
Always use fresh tips and pipettes for each solution and sample.
Maintain a clean work area.
Properly label all reagents and samples.
Store chemicals and reagents appropriately (freezer or fridge).
Do not collect samples if ill.
Properly dispose of waste.
Inform instructor on accidental chemical spills or concerns.
Wash hands thoroughly after lab work.

Preparation of Solutions

Molar solutions contain a specific number of grams of a solute per liter of solution.
Percentage solutions involve a specific weight of solute per 100ml of solution (w/v).
X Solutions are concentrated solutions that need to be diluted for testing.

Genomic DNA Extraction

Four main steps for extracting DNA from cells include cell lysis, lipids and proteins degradation, DNA purification, and ethanol precipitation.
Cell lysis: breaking down cell membranes to expose DNA.
Detergents and surfactants break down cell membranes and nucleus, while protease breaks down proteins.
Other processes, such as the removal of RNA using RNase are optional.
Centrifugation separates cellular debris from DNA.
Ethanol precipitation helps to form a DNA pellet, improving efficiency.
Common chemicals: Sodium chloride, tris-HCl, EDTA, detergents, proteins, salts, reagents for cell lysis, ethanol, and isopropanol.

Experimental Protocols (Example)

Samples such as blood are mixed with lysis solutions (example).
Samples are incubated at 56°C.
Ethanol is added to the solution, and the mixture is transferred to a column.
Several wash steps are performed.
DNA is eluted (released) using a buffer solution.

DNA concentration

DNA concentration is estimated using absorbance at 260nm, adjusting for turbidity.
A260/A280 ratio is approximately 1.8 for dsDNA. Ratios below 1.7 suggest protein contamination.
Turbidity, measured at 320nm, from proteins and organic compounds may affect results.

DNA Storage

EDTA is a heavy metal chelator, preventing DNA damage.
Storage temperatures are -70°C or 5°C (for short-term).
DNA is best stored with high salt (>1M), EDTA (>10mM) and low pH (8.5).
Ethidium bromide in CsCl is used for long-term storage, but in the dark.

Applications

Scientific: e.g., gene cloning, DNA analysis in cells and organisms.
Medicine: diagnosis of diseases, determination of virulence of microorganisms.
Forensics: identification of individuals in cases such as rape, petty theft, accidents, war victims, and paternity determination.

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Description

This quiz covers key concepts and techniques within the field of molecular diagnostics, including its application in personalized medicine, cancer treatment, and family planning. Test your knowledge on the role of companion diagnostics and laboratory procedures related to molecular testing.