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?

High concentrations of impurities and inhibitors Signup and view all the answers

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

Cell cultures Signup and view all the answers

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

Purity of extracted DNA Signup and view all the answers

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

Easier to lyse and obtain high yields of DNA Signup and view all the answers

Which statement is true regarding environmental samples in DNA extraction?

They may contain DNA from multiple species. Signup and view all the answers

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

To amplify a specific DNA sequence Signup and view all the answers

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

Denaturation Signup and view all the answers

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

It doubles Signup and view all the answers

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

Kary Mullis Signup and view all the answers

What technique is typically used to visualize DNA after extraction?

Agarose Gel Electrophoresis Signup and view all the answers

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

It is a safer alternative. Signup and view all the answers

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

It contains multiple phenyl groups. Signup and view all the answers

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

Alkali lysis. Signup and view all the answers

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

To increase the viscosity of the sample. Signup and view all the answers

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

45 minutes at 100 v. Signup and view all the answers

Which type of DNA structure is indicated as being supercoiled?

Closed circular DNA. Signup and view all the answers

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

5975 bp. Signup and view all the answers

Which of the following accurately describes DNA supercoiling?

It involves twisting of the circular DNA molecule. Signup and view all the answers

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

To ensure the recovery of DNA free of impurities Signup and view all the answers

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

It binds divalent ions that act as cofactors for nuclease Signup and view all the answers

What happens to proteins during the alkali lysis process?

They are denatured and lysed Signup and view all the answers

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

It precipitates denatured proteins and helps recover plasmid DNA Signup and view all the answers

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

Recombinant E. coli Signup and view all the answers

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

To select for transformed cells carrying the plasmid Signup and view all the answers

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

It facilitates the disruption of cell walls Signup and view all the answers

Which pH condition is typically achieved after the neutralization step?

Neutral pH Signup and view all the answers

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

To separate the DNA strands Signup and view all the answers

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

41°C Signup and view all the answers

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

Dideoxynucleoside triphosphates (ddNTPs) Signup and view all the answers

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

900 bp Signup and view all the answers

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

5 minutes Signup and view all the answers

What is the main function of DNA barcoding?

Identification of species using DNA Signup and view all the answers

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

To synthesize new DNA strands Signup and view all the answers

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

To determine the size of DNA fragments Signup and view all the answers

Which component is NOT required in the DNA sequencing reaction?

RNA polymerase Signup and view all the answers

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

Capillary gel electrophoresis Signup and view all the answers

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

High throughput capability Signup and view all the answers

What format can sequencing results be found in?

DNA sequence (FASTA, .txt) Signup and view all the answers

What does BLAST primarily do?

Find regions of local similarity between sequences Signup and view all the answers

What is one characteristic of third generation sequencing technologies?

Single molecule sequencing capability Signup and view all the answers

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

Petabyte-sized sequence data in public domain Signup and view all the answers

What is a feature of massive parallel sequencing?

Generates a lot of data Signup and view all the answers

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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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.