DNA Extraction and PCR Quiz
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Questions and Answers

What is the primary objective of DNA extraction?

  • To reduce the concentration of impurities
  • To isolate RNA from cellular components
  • To prepare samples for immediate analysis
  • To achieve high recovery of DNA with no impurities and inhibitors (correct)

Which of the following is NOT a source for DNA extraction listed?

  • Cell cultures
  • Plant tissue
  • Animal tissue
  • Mineral samples (correct)

Why might lysing plant and animal tissues be more challenging compared to other sources?

  • They contain more water than other samples
  • Their cell walls are harder to break down (correct)
  • They contain natural inhibitors that prevent extraction
  • They produce less DNA overall

What is a factor that could interfere with high recovery of DNA?

<p>High concentrations of impurities and inhibitors (A)</p> Signup and view all the answers

Which of the following samples may have the highest concentration of DNA for extraction?

<p>Cell cultures (C)</p> Signup and view all the answers

Which attribute is particularly important for downstream processes after DNA extraction?

<p>Purity of extracted DNA (C)</p> Signup and view all the answers

What characteristic of cell cultures makes them a preferred source for DNA extraction?

<p>Easier to lyse and obtain high yields of DNA (C)</p> Signup and view all the answers

Which statement is true regarding environmental samples in DNA extraction?

<p>They may contain DNA from multiple species. (D)</p> Signup and view all the answers

What is the primary purpose of the Polymerase Chain Reaction (PCR)?

<p>To amplify a specific DNA sequence (D)</p> Signup and view all the answers

Which of the following steps in PCR involves the separation of double-stranded DNA?

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

How does the amount of DNA change with each cycle of PCR?

<p>It doubles (A)</p> Signup and view all the answers

Who is credited with inventing PCR and winning the Nobel Prize for it?

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

What technique is typically used to visualize DNA after extraction?

<p>Agarose Gel Electrophoresis (D)</p> Signup and view all the answers

What is a primary advantage of using Gel Red over ethidium bromide?

<p>It is a safer alternative. (A)</p> Signup and view all the answers

Which of the following describes the IUPAC name provided for Gel Red?

<p>It contains multiple phenyl groups. (D)</p> Signup and view all the answers

Which technique was used to extract plasmid DNA in the provided content?

<p>Alkali lysis. (B)</p> Signup and view all the answers

What is the purpose of the gel loading dye when preparing samples for electrophoresis?

<p>To increase the viscosity of the sample. (C)</p> Signup and view all the answers

For how long and at what voltage was the agarose gel run in the experiment?

<p>45 minutes at 100 v. (C)</p> Signup and view all the answers

Which type of DNA structure is indicated as being supercoiled?

<p>Closed circular DNA. (D)</p> Signup and view all the answers

What is the molecular weight (MW) of the plasmid DNA mentioned?

<p>5975 bp. (B)</p> Signup and view all the answers

Which of the following accurately describes DNA supercoiling?

<p>It involves twisting of the circular DNA molecule. (A)</p> Signup and view all the answers

What is the primary objective of plasmid DNA extraction via alkali lysis?

<p>To ensure the recovery of DNA free of impurities (B)</p> Signup and view all the answers

In the alkali lysis method, what role does the chelator play?

<p>It binds divalent ions that act as cofactors for nuclease (B)</p> Signup and view all the answers

What happens to proteins during the alkali lysis process?

<p>They are denatured and lysed (D)</p> Signup and view all the answers

What is the result of the neutralization solution in the plasmid extraction process?

<p>It precipitates denatured proteins and helps recover plasmid DNA (B)</p> Signup and view all the answers

Which type of E. coli is primarily used in the process outlined?

<p>Recombinant E. coli (C)</p> Signup and view all the answers

Why is kanamycin used in the culture of E. coli during plasmid DNA extraction?

<p>To select for transformed cells carrying the plasmid (C)</p> Signup and view all the answers

What is the consequence of using an alkaline solution in the lysis step?

<p>It facilitates the disruption of cell walls (B)</p> Signup and view all the answers

Which pH condition is typically achieved after the neutralization step?

<p>Neutral pH (A)</p> Signup and view all the answers

What is the purpose of the initial denaturation step in PCR?

<p>To separate the DNA strands (A)</p> Signup and view all the answers

At what temperature does the annealing step occur during the PCR process?

<p>41°C (D)</p> Signup and view all the answers

Which component is essential for the chain-termination sequencing process?

<p>Dideoxynucleoside triphosphates (ddNTPs) (A)</p> Signup and view all the answers

What is the expected molecular weight of the PCR product when targeting the pET28a-sfGFP?

<p>900 bp (C)</p> Signup and view all the answers

For how long is the final extension step in the PCR cycling protocol conducted?

<p>5 minutes (D)</p> Signup and view all the answers

What is the main function of DNA barcoding?

<p>Identification of species using DNA (D)</p> Signup and view all the answers

In PCR, what is the primary role of the extension step?

<p>To synthesize new DNA strands (C)</p> Signup and view all the answers

What is the role of molecular weight markers in DNA analysis?

<p>To determine the size of DNA fragments (D)</p> Signup and view all the answers

Which component is NOT required in the DNA sequencing reaction?

<p>RNA polymerase (A)</p> Signup and view all the answers

What technology is used for separating DNA fragments in thermal cycle sequencing?

<p>Capillary gel electrophoresis (D)</p> Signup and view all the answers

What is the main advantage of Next Generation Sequencing (NGS) over previous methods?

<p>High throughput capability (C)</p> Signup and view all the answers

What format can sequencing results be found in?

<p>DNA sequence (FASTA, .txt) (D)</p> Signup and view all the answers

What does BLAST primarily do?

<p>Find regions of local similarity between sequences (C)</p> Signup and view all the answers

What is one characteristic of third generation sequencing technologies?

<p>Single molecule sequencing capability (B)</p> Signup and view all the answers

What does the International Nucleotide Sequence Database Collaboration (INSDC) primarily maintain?

<p>Petabyte-sized sequence data in public domain (B)</p> Signup and view all the answers

What is a feature of massive parallel sequencing?

<p>Generates a lot of data (D)</p> Signup and view all the answers

Flashcards

DNA Extraction

The process of isolating DNA from a sample, aiming for high purity and absence of impurities or inhibitors.

Cell cultures

A common source for DNA extraction, involving growing cells in a controlled environment.

Plant/animal tissue

Another source for DNA extraction, often from solid biological material.

Forensic evidence

DNA extraction from criminal or legal cases, often from trace samples.

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Environmental samples

DNA extraction from natural or ecological sources, like water or soil.

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Impurities and inhibitors

Substances that can affect DNA extraction quality, preventing downstream processing.

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Downstream processes

Further lab procedures that depend on high-quality DNA extraction

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High recovery of DNA

Extracting the maximum amount of DNA from the sample with the minimal impurities

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Plasmid DNA extraction

Process to isolate plasmid DNA for downstream use, free from impurities and inhibitors

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Alkali lysis

A step in plasmid DNA extraction that breaks open cells and denatures proteins

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Neutralization

Process to adjust pH to a suitable level for plasmid DNA precipitation.

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Plasmid DNA

Circular DNA molecule often found in bacteria, used for cloning DNA

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Escherichia coli

Bacterial species commonly used for DNA cloning and manipulation due to ease of handling

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Kanamycin

An antibiotic used to select for bacteria containing a plasmid

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Origin of replication

Specific DNA sequence where DNA replication begins

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LB Broth

A nutrient-rich broth used to grow bacteria

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Polymerase Chain Reaction (PCR)

A lab technique used to make many copies of a specific DNA segment.

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Thermal Cycling

Repeated heating and cooling steps in PCR to denature and anneal DNA.

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Denaturation

The step in PCR where the double-stranded DNA separates into two single strands.

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Annealing

The step in PCR where primers bind to the target DNA sequence.

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Elongation

The step in PCR where new DNA strands are synthesized from the primers.

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Gel Red 

A safer alternative dye to ethidium bromide, used to visualize nucleic acids (DNA and RNA) in gels.

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Agarose Gel Electrophoresis

A laboratory technique used to separate DNA, RNA, or proteins based on their size using an agarose gel.

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Supercoiled DNA

A tightly wound form of circular DNA, more compact than relaxed circular DNA.

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Molecular Weight (MW)

Measurement of size of a molecule, commonly used in the study of DNA to evaluate fragmentation.

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Electrophoresis

A technique used to separate molecules based on size and charge via an electric field.

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Ethidium Bromide

A DNA-binding dye, commonly used in the past to visualize DNA in gels. Now used less due to safety concerns.

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PCR Cycling

A series of temperature changes that repeat several times during PCR to amplify DNA. The steps include denaturation, annealing, and extension.

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Extension

The process where DNA polymerase extends the primers, creating a new strand of DNA complementary to the template strand.

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DNA Barcoding

A method of identifying organisms by analyzing short, standardized DNA sequences. It's like using a barcode to ID products.

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Sanger Sequencing

A method of determining the exact order of nucleotides in a DNA strand. It uses dideoxynucleotides (ddNTPs) to stop DNA synthesis at different points.

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Dideoxynucleotides (ddNTPs)

Special nucleotides that lack a 3'-hydroxyl group, preventing DNA polymerase from adding more nucleotides. This allows scientists to stop DNA synthesis at specific points.

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Chain-termination Sequencing

Another name for Sanger sequencing, highlighting the use of ddNTPs to terminate DNA synthesis during sequencing.

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ddNTPs

Modified nucleotides that lack a 3' hydroxyl group, preventing further DNA chain elongation. Each ddNTP is labeled with a different fluorescent marker, allowing for identification during sequencing.

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Chromatogram

A graphical representation of DNA sequence data, showing peaks corresponding to each base in the sequence.

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FASTA Format

A standard text-based format for representing DNA sequences, consisting of a single-line header followed by the actual sequence.

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NGS

Next Generation Sequencing - a high-throughput sequencing technique able to analyze millions or billions of DNA fragments simultaneously, providing massive amounts of data.

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Massive Parallel Sequencing

A key feature of NGS where millions or billions of DNA fragments are sequenced simultaneously, enabling rapid and high-throughput analysis.

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BLAST

A bioinformatics tool used to compare nucleotide or protein sequences to databases, identifying regions of similarity and inferring evolutionary relationships.

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INSDC

The International Nucleotide Sequence Database Collaboration, a global effort to share nucleotide sequence data in the public domain.

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Study Notes

DNA Extraction Workflow

  • Sample: Recombinant E. coli with pET28a-sfGFP
  • Bacterial Culture: Overnight culture of recombinant E. coli
  • Plasmid DNA Extraction: Using Alkali Lysis
  • Visualization: Agarose Gel Electrophoresis
  • Analysis: Spectrophotometric Analysis of Extracted DNA using UV Spectrophotometry
  • Amplification: Polymerase Chain Reaction (PCR) of sfGFP gene insert

Prokaryotic vs. Eukaryotic DNA

  • Prokaryotes (Bacteria and Archaea): No nucleus, circular, naked genomic DNA
  • Eukaryotes: Nucleus, linear genomic DNA organized as chromatin
  • Both have non-genomic DNA

DNA Extraction Objectives and General Steps

  • Objective: High DNA recovery, no impurities or inhibitors (for downstream processes)
  • General Steps:
    • Collection of cells
    • Cell lysis to release DNA
    • Separation of DNA from other cellular material
    • Precipitation and concentration of DNA

Plasmid DNA

  • Description: Extra-chromosomal DNA
  • Characteristics: Covalently closed, circular
  • Replication: Independent replication
  • Diagram: A circular molecule illustrated with various restriction enzyme sites, an origin, and antibiotic resistance genes (e.g., ampicillin)

Plasmid DNA Extraction via Alkali Lysis

  • Objective: Recovery of DNA for downstream processes, without inhibiting PCR
  • Solutions (I, II, III): Details on components required (e.g., glucose, TrisCl, EDTA, NaOH, SDS, potassium acetate, glacial acetic acid, ethanol, Tris, EDTA)

Recombinant Escherichia coli

  • Description: E. coli with introduced DNA
  • Culture: Cultured in LB Broth with Kanamycin

DNA Extraction: Next Steps

  • Visualization: Agarose gel electrophoresis
  • Quantitative/Qualitative Analysis: UV spectrophotometry
  • Downstream Applications: PCR, DNA sequencing, cloning for recombinant DNA applications

Electrophoresis

  • Technique: Migration of charged molecules through a porous gel in an electric field
  • Factors Affecting Migration Speed: Charge, shape, size of the molecule, gel concentration
  • Purpose: Molecular separation technique

Agarose Gel

  • Composition: Linear polysaccharide extracted from red marine algae
  • Components: D-galactose and 3,6-anhydro-L-galactose
  • Use: Used as a matrix for electrophoresis

DNA and RNA Migration in Agarose Gel

  • Porous Gel: 3D mesh filled with water
  • Strand Length and Gel Pore Size: Larger DNA fragments migrate slower through a dense gel (tight mesh) or a less dense gel (loose mesh)

Agarose Gel Electrophoresis (AGE)

  • Objective: Confirming success of genomic DNA extraction
  • Process: Agarose gel preparation, sample loading, electrophoresis, visualization

Buffer Systems

  • TAE (Tris-acetate-EDTA): Buffer for electrophoresis
  • TBE (Tris-borate-EDTA): Buffer for electrophoresis (usually faster)

Loading Buffers

  • Tracking dyes: Glycerol to increase density, dyes (e.g., xylene cyanol FF, bromophenol blue, orange G) with different migration rates
  • Purpose: Visualize movement of DNA fragments during electrophoresis

Visualizing DNA

  • Ethidium Bromide: Fluorescent dye used to visualize DNA fragments, but has known health risks
  • Gel Red: Safer alternative to ethidium bromide

DNA and UV Spectrophotometry

  • Nitrogenous Bases (DNA): Absorb light at 260 nm
  • Protein Backbone/Salts/Phenol: Absorb light at 230 nm
  • Amino Acids (aromatic rings): Absorb light at 280 nm
  • Absorption Graph: Illustrates relative absorption at different wavelengths

UV Spectrophotometry to Assess Purity and Concentration

  • Purity: DNA purity assessed by the ratio of absorbance at 260 nm (DNA) to 280 nm (protein); 1.8-1.9 for pure DNA, <1.8 for protein contamination, and >2.0 for RNA contamination.
  • Concentration: Calculate DNA concentration using absorbance values at 260 nm. Formula: [dsDNA] = (Abs260) x (dilution factor) x (50 µg/mL)

Polymerase Chain Reaction (PCR)

  • Overall Objective: To amplify the superfolder GFP gene insert within the given plasmid.
  • In Vitro Cloning: Cloning using heat-stable DNA polymerase
  • Thermal Cycling: Repeated cycles of denaturation, annealing, and extension of primers

PCR Protocol

  • Steps: Get reagents, prepare mix, set up conditions, analyze gel, negative/positive results & error analysis

NGS (Next-Generation Sequencing)

  • High-Throughput: High volume sequencing with micro/nano technologies
  • Different Technologies: Multiple from different companies
  • Applications: DNA and RNA libraries, shotgun and parallel sequencing, massive parallel sequencing

Information Databases (Public Domain)

  • International Nucleotide Sequence Database Collaboration (INSDC): Core infrastructure for sharing nucleotide sequence data.
  • Data Size: >9 petabytes in 2020

BLAST (Basic Local Alignment Search Tool)

  • Use: To find regions of similarity between sequences in public databases.

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Bio 140.02 Workflow Module 3 plasmid DNA postlab PDF

Description

Test your knowledge on DNA extraction processes and the principles of Polymerase Chain Reaction (PCR). This quiz explores the challenges, techniques, and important attributes related to DNA handling and analysis. Perfect for students familiar with molecular biology concepts.

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