DNA Isolation Methods and Structure 4

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Questions and Answers

What is the primary purpose of the swab taken from the inner cheek epithelium?

  • To isolate and purify DNA from epithelial cells (correct)
  • To assess oral hygiene and health
  • To perform a genetic test on cheek cells
  • To collect saliva for biochemical analysis

Which equipment is essential for the DNA isolation procedure from epithelial cells?

  • Thermal block and vortex
  • GeneMATRIX Swab-Extract DNA Purification Kit and laboratory centrifuge (correct)
  • Heating block and microtome
  • Spectrophotometer and microscope

What should be avoided prior to taking the swab for optimal cell collection?

  • Rinsing the mouth with water
  • Reading or studying
  • Drinking and eating (correct)
  • Using mouthwash

What is the first step in the DNA isolation procedure using the mini-column method?

<p>Saturating the membranes with activation buffer (D)</p> Signup and view all the answers

How long should each cheek be rubbed to optimize the amount of epithelial cells collected?

<p>30-40 seconds for each cheek using different sides of the cotton tip (D)</p> Signup and view all the answers

What is the main role of DNA in living organisms?

<p>To serve as the carrier of genetic information (B)</p> Signup and view all the answers

What type of bond connects nucleotides in a DNA strand?

<p>3',5'-phosphodiester bonds (D)</p> Signup and view all the answers

Which nitrogenous base pairs with thymine in DNA?

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

Which statement about the structure of DNA is correct?

<p>DNA has a right-handed double helix structure. (B)</p> Signup and view all the answers

Why are G-C bonds considered stronger than A-T bonds?

<p>They form three hydrogen bonds. (C)</p> Signup and view all the answers

What feature allows one DNA strand to be used for replication or repair of the other strand?

<p>Complementary base pairing (B)</p> Signup and view all the answers

Which component of nucleotides is found in DNA?

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

What is primarily required for the initial process of isolating DNA from biological materials?

<p>Specific isolation methods (B)</p> Signup and view all the answers

Which of the following is NOT a common source for DNA extraction in human studies?

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

Which stage does NOT take place in the process of DNA isolation?

<p>Nucleoprotein synthesis (C)</p> Signup and view all the answers

What type of preparation is the goal of DNA isolation?

<p>High-molecular-weight preparation (A)</p> Signup and view all the answers

Which method of DNA isolation would be most appropriate when working with amniotic fluid cells?

<p>Genomic DNA extraction (D)</p> Signup and view all the answers

What is the purpose of using an EDTA tube for blood collection in DNA isolation?

<p>Preventing DNA degradation (A)</p> Signup and view all the answers

For which of the following purposes is DNA isolation specifically advantageous?

<p>Producing next-generation drugs (A)</p> Signup and view all the answers

Which of the following factors does NOT influence the choice of DNA isolation method?

<p>Cost of reagents (D)</p> Signup and view all the answers

What is the primary aim of the purification stage in DNA isolation?

<p>To remove other cellular components (B)</p> Signup and view all the answers

What is the significance of the A260/A280 ratio in nucleic acid preparation?

<p>It assesses the purity of DNA or RNA preparations. (B)</p> Signup and view all the answers

At what wavelength does DNA exhibit its maximum absorbance?

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

What A260/A280 ratio value indicates significant protein contamination?

<p>1.5 (B)</p> Signup and view all the answers

Which method is NOT mentioned as a DNA isolation technique?

<p>DNA microarray analysis (B)</p> Signup and view all the answers

What is the role of proteinase K in DNA isolation?

<p>To degrade proteins during cell lysis. (C)</p> Signup and view all the answers

How should isolated DNA be ideally stored for longevity?

<p>At -20 °C (D)</p> Signup and view all the answers

Which component absorbs UV light at 280 nm, indicating protein presence?

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

What does an A260/A280 ratio of approximately 1.8 suggest about a sample?

<p>The sample contains pure double-stranded DNA. (D)</p> Signup and view all the answers

Which layer of the obtained lysate contains nucleic acids after extraction?

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

Which substance is used primarily for the extraction of DNA through organic methods?

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

What is the function of proteinase K in DNA isolation?

<p>To degrade DNA-binding proteins and cellular debris (D)</p> Signup and view all the answers

Which of the following aspects is NOT altered during the cell lysis process?

<p>Nucleic acid stability (B)</p> Signup and view all the answers

Which compound is commonly used to precipitate DNA during isolation?

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

During the column-based DNA isolation, what happens to the unbound impurities?

<p>They remain in the filtrate (A)</p> Signup and view all the answers

In magnetic DNA isolation, what is the initial action taken in the procedure?

<p>Lysis of cells with proteinase K (D)</p> Signup and view all the answers

What type of buffer is typically used for elution in DNA isolation procedures?

<p>Low-salt buffer (A)</p> Signup and view all the answers

Which layer of the lysate contains denatured proteins after extraction with phenol and chloroform?

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

What is the purpose of the washing steps in column-based DNA isolation?

<p>To remove impurities from the column (B)</p> Signup and view all the answers

What role do sodium ions play in the DNA isolation process?

<p>They promote DNA precipitation (C)</p> Signup and view all the answers

Which step follows after the DNA is bound to the magnetic beads?

<p>Washing to remove impurities (A)</p> Signup and view all the answers

Flashcards

DNA Composition

DNA is made of nucleotides, each containing a nitrogenous base, sugar, and phosphate.

Nucleotides

Building blocks of DNA, composed of a base, deoxyribose, and phosphate group.

Purines and Pyrimidines

Adenine and guanine are purines; cytosine and thymine are pyrimidines.

DNA Isolation

The process of extracting DNA from biological material for study or use.

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Applications of Isolated DNA

Isolated DNA can be used for detecting genetic diseases, drug production, and more.

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3',5'-Phosphodiester Bonds

Covalent bonds linking nucleotides in a DNA strand through the phosphate and sugar.

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Complementary Base Pairing

Adenine pairs with thymine, and guanine pairs with cytosine in DNA.

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Types of Nucleic Acids

Different forms of nucleic acids include genomic DNA, mitochondrial DNA, and plasmid DNA.

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Sample Sources for DNA

Common sources include blood, epithelial cells, and hair follicles.

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Antiparallel Strands

The two strands of DNA run in opposite directions, crucial for replication.

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Hydrogen Bonds in DNA

A-T pairs have 2 hydrogen bonds, G-C pairs have 3; G-C bonds are stronger.

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EDTA Role

EDTA prevents blood clotting and inhibits DNA-degrading enzymes.

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Stages of DNA Isolation

Stages include preparation, lysis, purification, and concentration of DNA.

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Cell Lysis

The process of breaking down cell membranes to release DNA.

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Purification in DNA Isolation

Separation of DNA from proteins and other cellular components.

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DNA Extraction from Blood

DNA is most commonly extracted from peripheral blood collected in EDTA tubes.

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Purity of DNA

Determined by the A260/A280 ratio of light absorbance.

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A260/A280 ratio

The ratio that indicates the purity of DNA or RNA preparations.

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Optical density (OD)

A measure of light absorption used in quantifying nucleic acids.

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Maximum absorbance for DNA

DNA absorbs UV light maximally at 260 nm.

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Protein's maximum absorbance

Proteins exhibit maximum absorbance at 280 nm.

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Contaminant absorbance

Organic compounds absorb UV light between 230-240 nm.

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DNA concentration calculation

C = (Cb * Ab) / Ast, where Cb is sample concentration.

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DNA storage temperature

Isolated DNA should ideally be stored at -20 °C.

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Solvent-based DNA isolation

DNA extraction using phenol and chloroform.

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Cell lysis methods

A process using detergents to degrade cell membranes.

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Swab Technique

Using a sterile swab to collect epithelial cells from the inner cheek.

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Activation Buffer

A solution used to prepare mini-columns for DNA isolation.

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Epithelial Cells

Cells that line the inner surfaces of the body, including the cheeks.

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Purity Assessment

Evaluating the quality and cleanliness of isolated DNA.

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Lysate

A mixture obtained after cell lysis containing various cellular components.

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Phenol and Chloroform extraction

A method to separate lipids from the aqueous phase in lysate.

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Aqueous layer

The upper layer of lysate containing nucleic acids after extraction.

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DNA precipitation

The process of forming a DNA pellet using sodium ions and alcohol.

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Proteolysis

The breakdown of proteins, aiding in the isolation of DNA.

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Proteinase K

An enzyme that digests proteins and deactivates DNases during DNA isolation.

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GeneMATRIX column

A column used for the selective binding of DNA during isolation.

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Magnetic method

A DNA isolation technique using magnetic beads to bind and recover DNA.

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Elution

The process of recovering purified DNA using an elution buffer.

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Centifugation

A process involving spinning samples to separate components by density.

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Study Notes

DNA Isolation

  • DNA is a biopolymer, with nucleotides as monomers.
  • Nucleotides consist of a nitrogenous base (adenine, guanine, cytosine, or thymine), deoxyribose (a five-carbon sugar), and a phosphate residue.
  • Purines (adenine and guanine) are bicyclic, while pyrimidines (cytosine and thymine) are monocyclic.
  • DNA typically exists as a double helix, with strands running antiparallel.
  • Guanine pairs with cytosine, and adenine pairs with thymine.
  • Hydrogen bonds form between base pairs, holding the helix together.
  • G-C pairs are stronger (3 hydrogen bonds) than A-T pairs (2 hydrogen bonds).
  • The sequence of one DNA strand is complementary to the other.

DNA Isolation Methods

  • Researchers use various methods to isolate DNA from different materials (blood, tissues, etc.).
  • The resulting DNA needs to be of high molecular weight and purified from proteins and inhibitors.
  • Crucial factors influencing isolation method selection include nucleic acid type, source, and material type.

DNA Isolation Stages

  • Stage 1: Preparation of biological material (purification, fragmentation, homogenization, buffering).
  • Stage 2: Cell disintegration and lysis (releasing DNA).
  • Stage 3: Purification (separating DNA from cellular components).
  • Stage 4: Concentration (removing low-molecular impurities).
  • Stage 5: Quality and quantitative evaluation (e.g., spectrophotometry).

DNA Purity Evaluation

  • The A260/A280 ratio measures DNA purity.
  • A ratio of ~1.8 suggests pure double-stranded DNA.
  • Ratios below 1.8 indicate protein contamination; A ratio of 1.5 suggests approximately 50% protein contamination.
  • DNA concentration can be calculated using a standard formula (Cb = Cst * Ab / Ast).

Practical Part

  • Preparing a cheek swab for DNA extraction.
  • Activating mini-columns before extraction.
  • Procedure for cheek cell collection.
  • Specific buffer additions, mixing, and incubation times in the DNA isolation procedure.
  • Using a spectrophotometer for quality analysis, assessing purity and concentration; calculating expected yield (1-10µg DNA).

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