Molecular Biology: PCR and Electrophoresis Quiz
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Questions and Answers

What is the main purpose of PCR in molecular biology?

  • To convert DNA into mRNA
  • To sequence RNA directly
  • To isolate proteins
  • To exponentially amplify DNA (correct)

Which enzyme is primarily used in the PCR process?

  • Taq DNA Polymerase (correct)
  • RNA polymerase
  • DNA ligase
  • Reverse transcriptase

At what temperature does the denaturation phase of the PCR cycle occur?

  • 50-60 ºC
  • 37 ºC
  • 68-72 ºC
  • 94 ºC (correct)

Which of the following is NOT an application of PCR?

<p>Protein synthesis (A)</p> Signup and view all the answers

What is the typical temperature range for the annealing phase in PCR?

<p>50-60 ºC (D)</p> Signup and view all the answers

Who developed the PCR technique and won the Nobel Prize for it?

<p>Kary Mullis (D)</p> Signup and view all the answers

What is the role of primers in the PCR process?

<p>They provide a starting point for DNA synthesis (A)</p> Signup and view all the answers

Which application of PCR is specifically used for identifying genetic matching?

<p>Genotyping (D)</p> Signup and view all the answers

What is the primary reason for wearing protective eyewear when observing DNA on a transilluminator?

<p>To protect against UV light damage (B)</p> Signup and view all the answers

How does agarose concentration affect DNA fragment mobility?

<p>Higher concentrations allow resolution of small fragments (C)</p> Signup and view all the answers

What might happen if water is mistakenly used instead of buffer in gel electrophoresis?

<p>There will be no migration of DNA (A)</p> Signup and view all the answers

Which buffer is commonly utilized for duplex DNA fragment migration?

<p>TBE (B)</p> Signup and view all the answers

What happens to DNA migration if a high voltage is applied during electrophoresis?

<p>Migration accelerates (A)</p> Signup and view all the answers

Which statement is true regarding low versus high agarose concentrations?

<p>Low concentrations allow better resolution for larger fragments (C)</p> Signup and view all the answers

What could occur if a concentrated buffer, like a 10X solution, is used during electrophoresis?

<p>Heat generation that could melt the gel (D)</p> Signup and view all the answers

What additional step is necessary to quantify DNA using a spectrophotometer after a crude extraction?

<p>Diluting the sample (A)</p> Signup and view all the answers

What is the primary function of the proofreading activity of DNA polymerase?

<p>To correct nucleotide incorporation errors (A)</p> Signup and view all the answers

Which statement about Phusion High-Fidelity DNA Polymerase is accurate?

<p>It was engineered by fusing a non-specific dsDNA-binding domain. (A)</p> Signup and view all the answers

What is the significance of maintaining equal concentrations of each dNTP in PCR?

<p>It minimizes misincorporation levels. (C)</p> Signup and view all the answers

Which component in the PCR buffer helps promote primer annealing to the template DNA?

<p>Potassium (K+) from KCl (C)</p> Signup and view all the answers

What role do magnesium ions (Mg2+) play in the PCR process?

<p>They form complexes with dNTPs and primers. (D)</p> Signup and view all the answers

What pH range is typically suitable for the activity of DNA polymerase in a PCR buffer?

<p>8.0 to 9.5 (B)</p> Signup and view all the answers

Which PCR component can help enhance specificity by destabilizing weak hydrogen bonds?

<p>Ammonium sulphate (NH4SO4) (C)</p> Signup and view all the answers

Which of the following statements about Taq DNA polymerase is incorrect?

<p>It has a proofreading function. (B)</p> Signup and view all the answers

What is the primary method used for quantitating DNA using gel electrophoresis?

<p>Comparing fluorescence intensity with DNA standards (A)</p> Signup and view all the answers

Which wavelength of UV light is primarily used for measuring nucleic acid concentration?

<p>260 nm (D)</p> Signup and view all the answers

What indicates a pure DNA sample when using the OD260/OD280 ratio?

<p>1.8 (B)</p> Signup and view all the answers

What limitation does a regular Spectrophotometer have regarding DNA quantitation?

<p>It cannot quantitate low amounts of DNA (less than 250 ng/mL). (C)</p> Signup and view all the answers

What does an absorbance reading at 280 nm indicate when measuring DNA purity?

<p>Contamination by phenol or protein (A)</p> Signup and view all the answers

What is the absorbance range for accurate Spectrophotometer readings?

<p>0.1–1.0 (B)</p> Signup and view all the answers

What does the absorbance at 230 nm measure in a DNA sample?

<p>Contamination by carbohydrates or salts (D)</p> Signup and view all the answers

What is a significant advantage of using Nanodrop Spectrophotometers over regular ones?

<p>Higher sensitivity for low DNA concentrations (A)</p> Signup and view all the answers

What is the primary reason for using tracking dyes in gel electrophoresis?

<p>To allow visual monitoring of electrophoresis progress (A)</p> Signup and view all the answers

Which of the following statements is true regarding DNA fragments in agarose gel?

<p>DNA migration can be visually monitored using tracking dyes. (A)</p> Signup and view all the answers

What is a significant characteristic of ethidium bromide (EtBr)?

<p>It intercalates between DNA bases and emits fluorescence upon UV exposure. (C)</p> Signup and view all the answers

What is the role of the anode in gel electrophoresis?

<p>To attract negatively charged DNA fragments (B)</p> Signup and view all the answers

Why should ethidium bromide be handled with care in the laboratory?

<p>It is a known mutagen and requires gloves during handling. (C)</p> Signup and view all the answers

What feature of RedSafe makes it preferable over ethidium bromide?

<p>It is non-toxic and not mutagenic. (A)</p> Signup and view all the answers

What effect does DNA diffusion have on the observation of gel electrophoresis results?

<p>It can blur the bands over time, making results less clear. (D)</p> Signup and view all the answers

What is the purpose of the UV transilluminator in visualizing DNA?

<p>To visualize the stained DNA under UV light. (C)</p> Signup and view all the answers

What is the optimal concentration range of MgCl2 for standard PCR reactions?

<p>1-4 mM (C)</p> Signup and view all the answers

How does excess Mg2+ affect DNA synthesis fidelity?

<p>It stabilizes DNA double strands. (A)</p> Signup and view all the answers

What primer concentration range is typically recommended?

<p>0.1 to 1 µM (A)</p> Signup and view all the answers

What is a consequence of high primer concentrations during PCR?

<p>Increased likelihood of mis-priming (A)</p> Signup and view all the answers

Which condition is likely to improve the specificity of primer annealing?

<p>Low primer concentration and high temperature (B)</p> Signup and view all the answers

What contributes to the formation of primer-dimers?

<p>High primer concentration (B)</p> Signup and view all the answers

What is mis-priming in the context of DNA synthesis?

<p>Primers annealing to a non-complementary sequence (D)</p> Signup and view all the answers

What is a key characteristic of a well-designed PCR primer?

<p>It should be complementary to the coding strand. (B)</p> Signup and view all the answers

Flashcards

PCR (Polymerase Chain Reaction)

A technique used to amplify a specific DNA sequence exponentially. It involves denaturing DNA, annealing primers, and extending the primers with DNA polymerase.

Taq DNA Polymerase

The enzyme used in PCR that can withstand high temperatures to copy DNA strands. It was isolated from a thermophilic bacterium found in hot springs.

Denaturation

The process of separating double-stranded DNA into single strands by applying heat.

Primers

Short, single-stranded DNA sequences that bind to specific regions of the target DNA sequence during PCR.

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Annealing

The process in PCR where the primers bind to their complementary sequences on the template DNA strands.

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Extension

The process in PCR where Taq DNA polymerase extends the primers, creating new DNA strands complementary to the template.

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PCR Cycle

The steps of denaturation, annealing, and extension are repeated multiple times, leading to an exponential amplification of the target DNA sequence.

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Applications of PCR

The use of PCR in various fields such as research, medicine, and forensic science.

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DNA Migration

A negatively charged molecule that moves towards the positive anode during electrophoresis.

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Loading Buffer

A buffer solution added to DNA samples before electrophoresis to help visualize the progress of the separation and ensure the samples sink to the bottom of the wells.

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Tracking Dyes

Dye used in electrophoresis to track the migration of DNA fragments.

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Agarose Gel Electrophoresis

A common method used to separate DNA fragments based on their size.

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DNA Staining

A method of visualizing DNA fragments in a gel by staining with a fluorescent dye which binds to DNA and emits light when exposed to UV.

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Ethidium Bromide (EtBr)

A fluorescent dye that intercalates between DNA bases and emits red/orange fluorescence when exposed to UV light.

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RedSafe

A safer alternative to EtBr. This dye also binds to DNA and emits green fluorescence when exposed to UV.

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UV Transilluminator

A device that uses UV light to visualize DNA (or RNA) that has been stained with a fluorescent dye.

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Agarose Concentration and DNA Fragment Separation

The concentration of agarose in a gel affects its ability to separate DNA fragments. Higher concentrations (e.g., 1.5%) are better for resolving smaller fragments, while lower concentrations (e.g., 0.7%) are better for separating larger fragments.

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Electrophoresis Buffer and DNA Mobility

Electrophoresis buffers like TAE or TBE create a conductive environment for DNA migration. The ionic strength of the buffer affects the migration rate of DNA fragments.

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Voltage and DNA Migration Speed

The voltage applied during electrophoresis directly impacts the speed of DNA fragment migration. Higher voltage leads to faster migration.

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Why Use Gel Electrophoresis for Crude DNA Quantification?

In crude DNA extraction, genomic DNA is often contaminated with other molecules, such as proteins and RNA. These contaminants can interfere with spectrophotometer measurements making it inaccurate for quantifying the isolated genomic DNA.

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Additional Step for Spectrophotometer Quantification

The presence of other molecules, like proteins and RNA, in a crude DNA extract interferes with spectrophotometer readings. To accurately quantify DNA using a spectrophotometer, you need to remove these contaminants.

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Proofreading DNA polymerase

A type of DNA polymerase that can proofread its work, detecting and correcting errors during DNA synthesis.

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3’ to 5’ exonuclease activity

The ability of a polymerase to remove incorrectly incorporated nucleotides, increasing accuracy during DNA replication.

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Phusion DNA polymerase

A proofreading DNA polymerase known for its high accuracy and speed, often used for demanding PCR applications.

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dNTPs (deoxyribonucleoside triphosphates)

The building blocks of DNA, comprised of a deoxyribose sugar, a phosphate group, and one of four nitrogenous bases: adenine (A), cytosine (C), guanine (G), or thymine (T).

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Buffer (in PCR)

The chemical environment surrounding a DNA polymerase reaction, including pH and concentrations of ions.

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Magnesium ions (Mg2+)

A crucial component of PCR, forming complexes with dNTPs, primers, and DNA templates, facilitating polymerase activity.

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Ammonium sulfate (NH4SO4)

A key component in PCR that helps destabilize weak hydrogen bonds between primers and template DNA, increasing specificity.

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Importance of buffer choice in PCR

The concentration of MgCl2 in a PCR reaction can significantly affect the specificity of the reaction, illustrating the importance of buffer optimization.

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Magnesium ions (Mg2+) in PCR

Magnesium ions (Mg2+) are crucial for DNA polymerase activity during PCR. Optimal concentration is essential, as too much can lead to misincorporation of nucleotides and prevent proper DNA denaturation, while too little hinders PCR product yield.

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Primers in PCR

Short, synthetic oligonucleotides that bind to specific DNA sequences (targets) during PCR, enabling amplification.

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Primer concentration

The concentration of primers used in PCR can significantly affect reaction outcome. Too high a concentration can lead to non-specific amplification and formation of primer-dimers.

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Mis-priming

Non-specific binding of primers to DNA sequences that are not the intended target, leading to amplification of undesired products.

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Primer-dimers

A situation where primers bind to each other instead of the target DNA sequence, resulting in undesired products.

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Primer specificity

The ability of primers to bind accurately and specifically to their intended target sequence.

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Hairpin formation

A situation where primers can bind to themselves due to self-complementarity, leading to undesired products.

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Coding strand and Rev primer

The coding strand is a DNA sequence that has the same sequence as the mRNA molecule produced from it. The reverse complement (Rev) primer has a sequence that is the reverse and complementary to the coding strand sequence.

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DNA quantitation using gel electrophoresis

A method for estimating the concentration of DNA by visualizing the fluorescence emitted by DNA intercalating dyes under UV light, comparing it to known DNA standards. This method is less accurate but can be used for lower DNA amounts.

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DNA quantitation using spectrophotometry

A technique for determining DNA purity and concentration by measuring its absorbance of UV light at specific wavelengths.

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OD260/OD280 ratio

A ratio calculated by dividing the absorbance of a DNA sample at 260nm by its absorbance at 280nm, used to indicate the purity of the DNA sample.

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UV absorbance at 260nm

A specific wavelength of UV light (260nm) absorbed by nucleic acids, used to determine DNA concentration.

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UV absorbance at 280nm

A wavelength of UV light (280nm) absorbed by proteins and other contaminants in DNA samples.

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UV absorbance at 230nm

A wavelength of UV light (230nm) absorbed by carbohydrates and other contaminants in DNA samples.

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Nanodrop spectrophotometer

A specialized spectrophotometer that can measure low concentrations of DNA with small sample volumes.

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Comparing DNA band intensities to standards

A method to evaluate the DNA concentration using a standard series of DNA samples with known concentrations, comparing the band intensities of the unknown sample to the standards.

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Study Notes

Course Overview

  • Course: BIOD21 - Advanced Molecular Biology Lab
  • Instructor: Prof. Sonia Gazzarrini
  • Semester: Fall 2024

Goal 1: Learning Lab Techniques

  • Planning and conducting experiments
    • Experimental plan (lab protocols/handouts)
    • Workflow
  • Data acquisition, interpretation, and troubleshooting
    • Careful description of each experimental step
    • Data discussion
    • Preparation of high-quality figures
  • Data presentation and discussion
    • Written lab report (research paper format)
    • Oral presentation (similar to lab meeting or conference presentations)

Goal 2: Molecular Techniques for Gene Regulation

  • Transcriptional regulation in response to stress
    • RNA isolation from control and treatment (stress) plants
    • cDNA synthesis and RT-qPCR
  • Generation and characterization of mutants
    • Genome editing by CRISPR/Cas9
    • Phenotypic analysis of wild-type and mutants
  • Protein localization
    • Subcellular localization of proteins using a translational reporter (YFP fusion protein) and confocal microscopy
  • Gene cloning and sequencing
    • Cloning strategies (Gateway, Golden Gate)
    • Bacterial transformation, plasmid isolation, and colony screening by PCR
    • Sequencing data analysis

Goal 2: Bioinformatics Analysis

  • Navigating molecular biology databases and repositories (NCBI, TAIR)
    • Using web-based tools for DNA and protein sequence analysis, alignment, primer design, etc.
    • In silico gene expression analysis using available transcriptomic data (microarray and RNAseq).
    • Primer and guide-RNA design
    • In-silico PCR
  • Alberts et al. (2002) Molecular Biology of the Cell (NCBI Bookshelf)
    • Chapter 8, sections on "Studying Gene Expression and Function" and "Isolating, Cloning, and Sequencing DNA" (NCBI Bookshelf links provided)
  • Lodish et al. (2000) Molecular Cell Biology (NCBI Bookshelf)
    • Section 7 "DNA cloning with plasmid vectors" (NCBI Bookshelf link provided)
  • Thermo Fisher Scientific (technical resources and reference library)
  • Addgene (protocols and in-house videos on Quercus)

Overview of Weeks 1-5 Labs

  • Studying gene regulation (transcriptional regulation by abiotic stress) using the model organism Arabidopsis thaliana
  • In silico primer testing for qPCR
  • Using primers in genomic DNA using regular PCR (genomic DNA isolation and PCR)
  • Exposing WT plants to control and abiotic stress conditions and determining if CBF4 is transcriptionally regulated
  • In silico analyses of gene expression using available transcriptomic data (microarray and RNAseq) (Bioinformatics Lab 2)

Week 1: Bioinformatics Lab 1

  • DNA sequence analysis and in silico PCR (NCBI)
    • CBF4 genomic, cDNA, and CDS sequences
  • Sequence alignment (Clustal Omega)
  • Primer design and in silico PCR (Primer Blast)
  • Bioinformatics tools for sequence analysis, including checking for introns, exons, and UTRs.

Setting up PCR Reactions: Master Mix and Controls

  • Components of a PCR master mix
    • DNA
    • Enzyme (e.g., Taq polymerase)
    • dNTPs
    • Buffer (e.g., MgCl2)
    • Primers
  • Steps to prepare a master mix
    • Preparing a master mix with all components, and then adding each into a tube
  • Importance of negative and positive controls

PCR Cycle

  • Steps in the PCR cycle:
    • Denaturation
    • Annealing
    • Extension
  • Optimizing conditions for different types of DNA

Important Considerations

  • Primer specificity
    • Minimizing mis-priming: selecting primers to avoid regions of sequence similarity to other genes
    • Primer concentration
    • Primer length and GC content
    • Choosing primers with the correct Tm for annealing temperature
  • Primer-dimer and hairpin formation
  • Design of gene-specific primers

Genomic DNA Isolation and Analysis

  • Procedure for genomic DNA isolation:
    • Sample crushing
    • Lysis buffer treatment
    • Supernatant transfer
    • Precipitation in isopropanol
    • Ethanol wash
    • Resuspension in TE buffer
  • Gel electrophoresis for genomic DNA analysis:
    • Agarose gel preparation and running
    • Staining and visualization of DNA fragments

DNA and RNA Quantitation

  • Gel electrophoresis
  • Spectrophotometer
    • DNA concentration measurement using Nucleic acid intercalating dyes (uv illumination) and standards
  • Spectrophotometer-based quantitation of DNA
    • Use of absorbance at 260nm, 280nm, and 230nm
    • Calculating the ratio OD260/OD280
  • Limitations of methods

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Related Documents

Lecture 1-2 (DNA & PCR).pdf

Description

Test your knowledge on the Polymerase Chain Reaction (PCR) and gel electrophoresis in molecular biology. This quiz covers aspects such as the purpose of PCR, key enzymes involved, and the significance of agarose concentration in DNA fragment mobility. Perfect for students and enthusiasts looking to deepen their understanding of these essential techniques.

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