Molecular Biology Techniques Quiz

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Questions and Answers

What is the purpose of the melt curve step added after the PCR cycle when using SYBR green?

To stabilize the dsDNA during amplification.

To enhance the fluorescence intensity of SYBR green.

To reduce the impact of background fluorescence.

To overcome the non-specificity of SYBR green. (correct)

Which of the following accurately describes the TaqMan probe used in specific detection chemistries?

It uses a quencher located at the 5' end of the oligonucleotide probe.

It consists of a single dye that binds non-specifically to dsDNA.

It relies on the 5'-3' exonuclease activity of Taq polymerase. (correct)

It emits light without the involvement of a fluorophore.

Which of the following statements about SYBR green is true?

It binds to the minor groove of double-stranded DNA (dsDNA). (correct)

It specifically binds to the 5' end of the oligonucleotide probe.

It requires high temperatures to maintain its binding specificity.

The fluorescence intensity can only be detected during the annealing phase.

What role does the quencher play in a TaqMan probe setup?

It dissipates energy from the fluorophore in the form of light or heat. Signup and view all the answers

What happens to the TaqMan probe during hybridization to the complementary target sequence?

The quenching effect is relieved, allowing fluorescence to occur. Signup and view all the answers

What is the primary purpose of a microarray?

To detect the presence of viruses in samples Signup and view all the answers

What is hybridization in the context of microarrays?

The pairing of complementary DNA strands Signup and view all the answers

Which statement correctly describes CGI in gene expression?

Gene expression can differ based on the type of disease. Signup and view all the answers

What type of microarray contains antibodies to detect target antigens?

Antibody microarray Signup and view all the answers

Which type of blotting technique is used for the detection of RNA?

Northern blotting Signup and view all the answers

In Western blotting, what is the role of the protease inhibitor cocktail?

To prevent protein degradation Signup and view all the answers

What is the solid support in blotting techniques typically made of?

Nylon membrane Signup and view all the answers

What is the primary purpose of the fluorescent dye or enzyme such as horseradish peroxidase (HRP) in protein detection?

To generate a signal indicating the location of proteins Signup and view all the answers

Which chemical is used in DNA extraction during blotting techniques?

Detergent Signup and view all the answers

During Southern and Northern blotting, what is added to detect the target sequence?

A labeled nucleic acid with a complementary sequence Signup and view all the answers

In the context of protein detection, which substrate is associated with luminol?

Chmiluminescent substrate Signup and view all the answers

What is the first step in the PCR process as outlined above?

Initial Denaturation Signup and view all the answers

Which component in PCR is responsible for synthesizing new DNA strands?

DNA polymerase (Taq polymerase) Signup and view all the answers

What is a characteristic feature of the dot blot technique?

Samples are spotted onto a membrane without separation Signup and view all the answers

What is the typical detection limit for colorimetric substrates compared to chmiluminescent substrates in protein assays?

Colorimetric substrates detect in the nanogram range, chmiluminescent in femtogram Signup and view all the answers

Which ion is the most common cation used in PCR for DNA polymerase activity?

Mg2+ (Magnesium ion) Signup and view all the answers

What is the purpose of adding formaldehyde to the gel during RNA electrophoresis?

To denature RNA to ensure it runs properly in the gel Signup and view all the answers

Which of the following statements about nitrocellulose membranes is true?

They are brittle and not suitable for stripping and reprobing. Signup and view all the answers

What is the function of the blocking buffer in western blotting?

To prevent non-specific binding of antibodies Signup and view all the answers

Which method of transfer for proteins results in higher efficiency?

Wet transfer Signup and view all the answers

During which process are the separated fragments transferred from the gel to a solid support membrane?

Blotting Signup and view all the answers

What is the role of UV radiation in the immobilization step?

To permanently fix the fragments on the membrane Signup and view all the answers

Which form of gel electrophoresis is commonly used for the separation of protein fragments?

Polyacrylamide gel electrophoresis (PAGE) Signup and view all the answers

Which method of transferring proteins from a gel is characterized by using a small amount of buffer?

Semi-dry transfer Signup and view all the answers

What is the primary purpose of using high acrylamide concentration in gel electrophoresis?

To create a smaller mesh size for separating smaller proteins Signup and view all the answers

Which component initiates the polymerization of acrylamide and bisacrylamide?

Ammonium persulfate (APS) Signup and view all the answers

How do proteins migrate in the gel during electrophoresis?

Smaller proteins migrate faster as they are negatively charged Signup and view all the answers

What role does TEMED play in the polymerization process?

It splits persulfate ions into free radicals Signup and view all the answers

Which staining method is most sensitive in detecting proteins?

Silver stain Signup and view all the answers

What happens during the fixation step of protein staining?

An acid or alcohol wash limits diffusion of protein bands Signup and view all the answers

What characteristic change occurs when Coomassie dye binds to proteins?

The color changes to intense blue under acidic conditions Signup and view all the answers

Which statement is true regarding the distance migrated by proteins in the gel?

Distance migrated is inversely proportional to the molecular mass of the protein Signup and view all the answers

What is the primary purpose of using gel electrophoresis for analyzing virus components?

To compare the sizes of viral nucleic acids and capsid proteins Signup and view all the answers

Which gel type is better suited for separating large molecules?

Agarose Signup and view all the answers

What component in the denaturation buffer influences protein charge during Polyacrylamide Gel Electrophoresis?

Sodium dodecyl sulfate (SDS) Signup and view all the answers

Which statement is true regarding the toxicity of gel types used in electrophoresis?

Polyacrylamide is potent neuro-toxic and agarose is non-toxic Signup and view all the answers

What is the role of the stacking gel in electrophoresis?

To concentrate proteins before entering the separating gel Signup and view all the answers

Which staining method is commonly used for visualizing DNA in agarose gels?

Ethidium bromide Signup and view all the answers

How do proteins behave when migrating through the stacking gel?

They migrate at the same rate, forming a narrow band Signup and view all the answers

Which is a consequence of using 2-Mercaptoethanol in the denaturation buffer?

Prevents disulfide bonds from forming Signup and view all the answers

Signup and view all the answers

Study Notes

Gel Electrophoresis

Gel electrophoresis is a method used to separate and analyze macromolecules (nucleic acids and proteins) and their fragments based on molecular weight.
It is used to analyze virus components, as the molecular weights of viral capsid proteins and nucleic acids are distinctive features of viruses.

Gel Types

Agarose: A polysaccharide extracted from seaweed, used for large molecule separation, such as nucleic acids. It is non-toxic.
Polyacrylamide: A cross-linked polymer of acrylamide, used for smaller molecules. It is potent neuro-toxic. Nucleic acid and protein separation can be performed.

Gel Casting

Agarose gels are cast horizontally.
Polyacrylamide gels are cast vertically.

Toxicity

Agarose gels are non-toxic.
Polyacrylamide gels are potent neuro-toxic.

Separation

Agarose gels separate large molecules like nucleic acids.
Polyacrylamide gels separate smaller molecules like nucleic acids and proteins.

Staining

Staining methods, like ethidium bromide or RedSafe, are used to visualize DNA under UV light. These bind to DNA and cause fluorescence.
Staining can be done before or after running the gel. Post-run staining is common for protein separation.

Determination of Molecular Mass of Coat Proteins by Polyacrylamide Gel Electrophoresis (PAGE)

Steps: Denaturation involves purifying the virus and adding denaturation buffer to denature the viral capsid into polypeptide subunits.
Purpose:
- Denature the viral capsid into polypeptide subunits.

Electrophoresis (additional notes from page 2)

Buffers: Denaturation buffer may contain SDS (Sodium dodecyl sulfate) a detergent that gives proteins a net negative charge, leading to protein unfolding; and 2-ME (2-mercaptoethanol) a reducing agent to break disulfide bonds.
Stacking Gel: The concentration of acrylamide is lower (approximately 4%) in the stacking gel. This gel concentrates proteins while they migrate.
Resolving Gel: The lower resolving gel contains a higher concentration of acrylamide (approximately 12%), allowing separation of protein fragments based on size.

General Staining Steps (page 3)

Remove electrophoresis buffer (water wash).
Fix (Use acid or alcohol to prevent protein diffusion).
Wash.
Stain (Use a dye or chemical to bind to proteins).
Destain (remove excess dye). Examples include Coomassie dye stain (turns proteins blue) and silver stain (high sensitivity).

Microarray (page 4)

Microarrays are collections of thousands to millions of microscopic spots containing known nucleic acid fragments (probes or reporters) attached to a solid surface, called a 'chip'.
They are used for detecting the presence of viral samples, gene expression, and comparative gene hybridization (CGH).
Hybridization is the process of forming double-stranded DNA molecules through base pairing between complementary sequences.
Antibody microarrays use protein-specific capture antibodies on a solid surface to detect interaction with target antigens.

Blotting (page 5)

Blotting is a technique to transfer macromolecules (DNA, RNA, or proteins) from a gel to a solid support (e.g., nitrocellulose, PVDF) for further analysis.
Types of blotting include Southern (DNA), Northern (RNA), and Western (protein) blotting.
Sample Preparation: involves extracting and preparing proteins to visualize them further via techniques.
Gel Electrophoresis: PAGE or agarose gels are used based on the separation of the macromolecules.
Blotting (page 6): Various methods exist for transfer.
- Wet Transfer: uses a large amount of buffer; vertical transfer; and is slow. High efficiency (80-100%).
- Semi-Dry Transfer: uses a small amount of buffer; horizontal placement; and is faster. Slightly less efficient (60-80%)
Immobilization: Fixing the fragments onto the membrane. can be done through UV radiation or heat.

Hybridization (page 7)

Western Blotting: A primary antibody is incubated with the membrane overnight at 4°C. Then washing occurs to remove unbound antibody. Secondary antibody (labeled with a detectable signal, e.g., radioactively, fluorescent dye, or enzyme) is added, and further washing is performed. HRP (horseradish peroxidase) is often used as a signal.
Southern/Northern Blotting: Labelling DNA/RNA with a homologous sequence and detection using specific substrates are common.
Methods like colorimetric (using TMB, developing a purple-black color), or chemiluminescent substrates are used. These are different for specific use-cases.

Dot Blot (page 7)

A technique to detect and identify proteins, DNA, or RNA directly on a membrane without prior separation via electrophoresis.

PCR (Polymerase Chain Reaction (pages 8-9))

A method used to amplify a specific DNA sequence. It involves cycles of heating (denaturation) and cooling (annealing and extension).
Components: Template DNA, DNA polymerase (e.g., Taq polymerase), primers, nucleotides (dNTPs), buffer, and metal ions (e.g., Mg2+).
Steps: (initial denaturation, denaturization, annealing, extension/elongation)
Cycles/steps: are repeated to exponentially amplify the target DNA.

RT-PCR (Reverse Transcription PCR) (page 10)

Combines reverse transcription of RNA into cDNA followed by PCR amplification. This allows for studying gene expression levels.
Multiplex PCR: simultaneously detects multiple targets (genes) in a single reaction using different primer pairs for each target. The primer pairs need to be highly specific.

qPCR (Quantitative PCR) (page 11)

A real-time PCR technique combines DNA amplification and quantification of DNA.
Components: Real-time chemistries, and conventional PCR components.
Threshold Line: The level to detect signal when fluorescence intensity is above the background.
Ct value:
- The cycle number where the fluorescence of the PCR product crosses the threshold line.

Specific Probes (page 12)

TaqMan probes: consist of a fluorophore and quencher, which prevents fluorescence until the probe is cleaved. cleavage of the probe occurs after priming/elongation, which is measured.

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Description

Test your knowledge on key molecular biology techniques, including PCR, microarrays, and blotting methods. This quiz covers principles behind SYBR green, TaqMan probes, and the use of fluorescent dyes in protein detection. Ensure you understand hybridization and gene expression concepts as well.