Instruction 4 - DNA isolation
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Questions and Answers

What is a crucial goal when isolating DNA?

  • To obtain a high-molecular-weight preparation, purified from proteins and DNA enzyme inhibitors. (correct)
  • To ensure the DNA contains inhibitors.
  • To decrease the purity of the DNA.
  • To obtain low-molecular-weight preparation.

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

  • The material origin such as plant, animal, or bacterial.
  • The current weather conditions. (correct)
  • The expected results such as purity and isolation time.
  • The type of nucleic acid, such as genomic or mitochondrial DNA.

    • Why is EDTA used in blood collection tubes for human DNA studies?

  • To prevent blood clotting and inhibit deoxyribonucleases. (correct)
  • To increase the degradation of DNA.
  • To promote blood clotting.
  • To activate deoxyribonucleases.

    • Which of these sources is LEAST commonly used for DNA extraction in diagnostic studies?

    <p>Blood spots.</p> Signup and view all the answers

    In the process of isolating DNA, what occurs during the 'cell disintegration and lysis' stage?

    <p>Release of DNA into a solution and protection from degradation.</p> Signup and view all the answers

    What is typically the final stage after the concentration of a DNA preparation?

    <p>Removal of all low-molecular-weight impurities.</p> Signup and view all the answers

    Why must 'preliminary preparation of the biological material for DNA isolation' be performed?

    <p>To prepare the material through purification, fragmentation, homogenization, and suspension in preparation for lysis.</p> Signup and view all the answers

    What is the role of the stage that separates the nucleic acid from other cellular components?

    <p>It isolates the DNA from other parts of the cells.</p> Signup and view all the answers

    At what wavelength does DNA exhibit maximum absorbance?

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

    What does an A260/A280 ratio of approximately 1.8 indicate?

    <p>Pure dsDNA.</p> Signup and view all the answers

    What does an A260/A280 ratio of 1.5 suggest?

    <p>Protein contamination of around 50%.</p> Signup and view all the answers

    Which of these is NOT a method for DNA Isolation described in the text?

    <p>Electrophoresis using agarose gel</p> Signup and view all the answers

    Which of these is NOT a detergent used in the DNA isolation process?

    <p>Proteinase K</p> Signup and view all the answers

    What is the role of proteinase K in DNA isolation?

    <p>To degrade proteins.</p> Signup and view all the answers

    If a DNA sample has an absorbance of 0.5 at 260nm and the standard with a concentration of 100ng/uL has an absorbance of 0.2 at 260nm, what is the concentration of the analyzed sample?

    <p>250 ng/uL</p> Signup and view all the answers

    Why might a DNA sample have an A260/A280 ratio below 1.8?

    <p>Contamination by proteins.</p> Signup and view all the answers

    In DNA, which type of bond connects the nitrogenous base to the deoxyribose sugar?

    <p>N-glycosidic bond</p> Signup and view all the answers

    Which of the following describes the arrangement of DNA strands in a double helix?

    <p>Antiparallel, with one strand running 5' to 3' and the other 3' to 5'</p> Signup and view all the answers

    Which of the following best describes the chemical nature of guanine and adenine?

    <p>Both are purines</p> Signup and view all the answers

    What type of bond is responsible for linking successive nucleotides in a DNA strand?

    <p>Phosphodiester bond</p> Signup and view all the answers

    How many hydrogen bonds are formed between a guanine-cytosine base pair?

    <p>Three</p> Signup and view all the answers

    Which statement correctly describes the location of the nitrogenous bases and the sugar-phosphate backbone in the DNA double helix?

    <p>Nitrogenous bases are on the inside and the sugar-phosphate is on the outside</p> Signup and view all the answers

    If one strand of a DNA molecule has the sequence 5'-ATGC-3', what would be the sequence of the complementary strand?

    <p>3'-TACG-5'</p> Signup and view all the answers

    What property of DNA allows one strand to serve as a template for the synthesis or repair of the other strand?

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

    What is the primary purpose of activating the mini-column with Buffer S before adding the cell lysate?

    <p>To saturate the membrane and prepare it for DNA binding.</p> Signup and view all the answers

    What is the recommended time frame to avoid eating, drinking, smoking, or brushing teeth before collecting a cheek swab?

    <p>Approximately 1-2 hours.</p> Signup and view all the answers

    During the cheek cell collection procedure, what is the approximate recommended time for rubbing each cheek?

    <p>30-40 seconds</p> Signup and view all the answers

    In the context of this procedure, what does the term 'DNase-free' mean?

    <p>The tubes and tips are free from enzymes that can degrade DNA.</p> Signup and view all the answers

    According to the protocol, what is the correct order of steps after opening the sterile swab package?

    <p>Rub the cheeks, insert into tube, snap the stick, then close the tube.</p> Signup and view all the answers

    What is the main purpose of the Vortex during the described procedure?

    <p>To mix the samples thoroughly.</p> Signup and view all the answers

    Which piece of equipment is used to quantify and assess the purity of isolated DNA during the experiment?

    <p>Spectrophotometer</p> Signup and view all the answers

    What type of tube is recommended for inserting the swab after cheek cell collection?

    <p>2 ml DNase-free tube with a rounded bottom.</p> Signup and view all the answers

    During the phenol-chloroform extraction method, which layer contains the nucleic acids?

    <p>The upper aqueous layer</p> Signup and view all the answers

    What is the primary purpose of Proteinase K during DNA isolation?

    <p>To deactivate DNases and digest proteins, including DNA-binding proteins</p> Signup and view all the answers

    In column-based DNA isolation, what is the purpose of adding buffers and ethanol after Proteinase K treatment?

    <p>To create conditions for selective DNA binding to the column</p> Signup and view all the answers

    During column-based DNA isolation, how is the purified DNA eluted from the column?

    <p>Using a low-salt buffer, such as Tris-HCl, TE, or distilled water</p> Signup and view all the answers

    What is the function of magnetic beads in the magnetic method of DNA isolation?

    <p>To bind DNA molecules for easy separation</p> Signup and view all the answers

    What is removed during the washing steps in both magnetic and column-based DNA isolation methods?

    <p>Impurities, such as cellular debris and unbound molecules</p> Signup and view all the answers

    What is the purpose of sodium ions in DNA precipitation during the phenol-chloroform extraction method?

    <p>To neutralize the negative charge of the DNA backbone and facilitate aggregation</p> Signup and view all the answers

    How is cell lysis achieved in both column-based and magnetic DNA isolation methods?

    <p>By adding a lysis buffer and Proteinase K</p> Signup and view all the answers

    What is the purpose of adding Proteinase K during the DNA isolation procedure?

    <p>To degrade proteins</p> Signup and view all the answers

    What is the function of the Wash SX1 buffer in the DNA isolation process?

    <p>To clean the spin-column of impurities</p> Signup and view all the answers

    During which step is the lysate centrifuged at 11 000 x g for 1 minute?

    <p>After transferring lysate to the spin-column</p> Signup and view all the answers

    What is the correct incubation temperature after mixing with Lyse S buffer?

    <p>56°C</p> Signup and view all the answers

    What is the expected yield of DNA isolation from cheek epithelial cells?

    <p>1 to 10 µg</p> Signup and view all the answers

    What ratio indicates a good purity of isolated DNA when using a spectrophotometer?

    <p>A260/A280 ratio of 1.8</p> Signup and view all the answers

    What should be done to avoid transferring traces of DNA between spin-columns?

    <p>Use a new micropipette tip for each step</p> Signup and view all the answers

    What is the role of the Elution buffer in the DNA isolation process?

    <p>To elute the bound DNA from the spin-column</p> Signup and view all the answers

    What type of biological material is commonly used for DNA isolation in humans?

    <p>Peripheral blood</p> Signup and view all the answers

    Which factor is NOT considered when choosing a DNA isolation method?

    <p>Color of the biological material</p> Signup and view all the answers

    What is the function of EDTA in blood collection tubes for DNA studies?

    <p>To inhibit deoxyribonucleases</p> Signup and view all the answers

    During which stage of DNA isolation is the cell lysis achieved?

    <p>Cell disintegration and lysis</p> Signup and view all the answers

    What type of cells are least commonly used for DNA extraction in diagnostic studies?

    <p>Fine needle biopsy tissue fragments</p> Signup and view all the answers

    Which stage in DNA isolation involves separating nucleic acids from cellular components?

    <p>Purification stage</p> Signup and view all the answers

    Which of the following is a reason for the initial preparation of biological material before DNA isolation?

    <p>To ensure DNA is soluble and protected</p> Signup and view all the answers

    What is the end goal of DNA isolation procedures?

    <p>To obtain a high-quality DNA preparation</p> Signup and view all the answers

    What type of bond specifically links the nitrogenous bases to the deoxyribose sugar in DNA?

    <p>N-glycosidic bond</p> Signup and view all the answers

    How many hydrogen bonds are formed between adenine and thymine in a DNA double helix?

    <p>Two</p> Signup and view all the answers

    What structural feature maintains the stability of the DNA double helix?

    <p>Hydrogen bonds between bases</p> Signup and view all the answers

    Which pair of nitrogenous bases forms three hydrogen bonds in DNA?

    <p>Guanine and Cytosine</p> Signup and view all the answers

    What kind of nucleotides does DNA contain?

    <p>A mixture of purines and pyrimidines</p> Signup and view all the answers

    What is the significance of complementary base pairing in DNA?

    <p>It enables DNA replication and repair.</p> Signup and view all the answers

    Which sugar is found in the backbone of DNA?

    <p>Deoxyribose</p> Signup and view all the answers

    What characteristic of DNA allows it to form a double helix structure?

    <p>The antiparallel orientation of strands</p> Signup and view all the answers

    What is the significance of the A260/A280 ratio in evaluating DNA purity?

    <p>A ratio below 1.8 suggests significant protein presence.</p> Signup and view all the answers

    Which wavelength is associated with maximum absorbance for nucleic acids?

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

    What does an A260/A280 ratio of 1.5 indicate?

    <p>Presence of roughly 50% protein.</p> Signup and view all the answers

    In DNA isolation, what is the primary purpose of using detergents during cell lysis?

    <p>To disrupt cell membranes and denature proteins.</p> Signup and view all the answers

    How is the concentration of DNA calculated using absorbance measurements?

    <p>Using Cb = (Ab / Ast) * Cst.</p> Signup and view all the answers

    Which method is used to isolate DNA by binding it to a carrier?

    <p>Silica column chromatography.</p> Signup and view all the answers

    What is the ideal storage temperature for isolated DNA for extended periods?

    <p>-20 °C.</p> Signup and view all the answers

    What role does Proteinase K serve in the DNA isolation process?

    <p>To degrade proteins and facilitate lysis.</p> Signup and view all the answers

    What is the primary purpose of saturating the mini-column with activation buffer before adding the lysate?

    <p>To facilitate the binding of DNA to the mini-column</p> Signup and view all the answers

    During the cheek cell collection, what is the recommended maximum duration for rubbing each cheek?

    <p>30-40 seconds</p> Signup and view all the answers

    What is the significance of using DNase-free equipment during the DNA isolation process?

    <p>To inhibit the degradation of DNA by nucleases</p> Signup and view all the answers

    Which of the following actions should NOT be taken to ensure effective collection of cheek epithelial cells?

    <p>Brush teeth immediately before the procedure</p> Signup and view all the answers

    What is the purpose of the vortex in the DNA isolation procedure?

    <p>To evenly distribute the DNA within the buffer solution</p> Signup and view all the answers

    What type of tube is recommended for collecting epithelial cells after using the swab?

    <p>2 ml DNase-free tube with a rounded bottom</p> Signup and view all the answers

    What precautions should be taken before performing the DNA isolation from cheek cells?

    <p>Avoid eating, drinking, and smoking for 1-2 hours</p> Signup and view all the answers

    What will likely happen if the cotton tip of the swab is touched with fingers before sample collection?

    <p>Contamination with skin cells</p> Signup and view all the answers

    What is the purpose of adding 400 μl of Lyse S buffer and 10 μl of Proteinase K at the start of the DNA isolation procedure?

    <p>To lyse cells and degrade proteins</p> Signup and view all the answers

    Which step involves centrifuging the lysate at 11 000 x g for 1 minute?

    <p>After transferring the lysate to the DNA binding spin-column</p> Signup and view all the answers

    What is the expected yield of isolated DNA from cheek epithelial cells after the isolation process?

    <p>1 to 10 µg DNA</p> Signup and view all the answers

    What should be avoided when transferring the Elution buffer to the spin-column?

    <p>Touching the column walls with the micropipette</p> Signup and view all the answers

    At what temperature should the mixture with Lyse S buffer be incubated for optimal results?

    <p>56°C</p> Signup and view all the answers

    What does a spectrophotometer measure in the context of DNA isolation?

    <p>Concentration and quality of the isolated DNA</p> Signup and view all the answers

    What is the next step after adding 500 μl of Wash SX1 buffer to the spin-column?

    <p>Centrifuge for 1 minute at 11 000 x g</p> Signup and view all the answers

    What is the primary role of the phenol and chloroform mixture during DNA isolation?

    <p>To extract lipids from the aqueous phase, separating them from nucleic acids</p> Signup and view all the answers

    How is DNA immobilized during the magnetic method of DNA isolation?

    <p>By binding to magnetic beads that are then exposed to a magnet</p> Signup and view all the answers

    What is the purpose of Proteinase K during the DNA isolation procedure?

    <p>To digest proteins, including DNA-binding proteins, and break down cellular debris</p> Signup and view all the answers

    Which of the following steps is NOT involved in the phenol-chloroform extraction method of DNA isolation?

    <p>Binding DNA to magnetic beads</p> Signup and view all the answers

    How is the purified DNA eluted from the column during column-based DNA isolation?

    <p>By washing the column with a low-salt buffer, typically containing Tris-HCl, TE, or distilled water.</p> Signup and view all the answers

    Why is it important to prevent contamination of the DNA sample during isolation?

    <p>All of the above</p> Signup and view all the answers

    Study Notes

    DNA Isolation

    • DNA is found in cell nuclei, forming chromosomes, carrying genetic information for living organisms.
    • DNA is a linear biopolymer, with nucleotides as monomers.
    • Nucleotides consist of a nitrogenous base (adenine, guanine, cytosine, or thymine), deoxyribose sugar, and a phosphate residue.
    • Adenine and guanine are purines (bicyclic), while cytosine and thymine are pyrimidines (monocyclic).
    • DNA forms a double helix, antiparallel strands, with base pairs (guanine-cytosine and adenine-thymine) held together by hydrogen bonds.
    • G-C bonds are stronger than A-T bonds (three vs. two hydrogen bonds).
    • DNA sequence is complementary; one strand can be used for replication/repairing the other in case of damage.
    • DNA isolation aims to obtain high molecular-weight, protein-free DNA.

    DNA Research Applications

    • DNA isolation is crucial in many fields, including medical biology/diagnosticians.
    • Isolated DNA is used to detect genetic diseases, assess cancer risk, develop drugs, and do cell/genetic engineering.
    • Detecting various genetic diseases, assessing predisposition to tumors, and producing next-generation drugs are uses of DNA.

    DNA Isolation Procedures

    • Various methods exist; the best choice depends on material type (plant, animal, bacterial, viral, etc.) and sample (tissue, organs, cells).
    • Stages include sample preparation (purification, fragmentation, homogenization, suspension), cell lysis (releasing DNA), DNA purification (removing cellular components), concentration (removing low-molecular-weight impurities), and quality assessment.

    DNA Purity Assessment

    • DNA purity is assessed by measuring UV light absorption using spectrophotometry at 260 and 280 nanometers (nm).
    • A 260/280 ratio of 1.8 is ideal for pure double-stranded DNA (dsDNA).
    • Absorbance at 260 nm is used to calculate DNA concentration.
    • 230-240 nm is also used for quality assessment; contaminants may also absorb in this range.

    DNA Isolation Methods

    • There are solvent-based (phenol/chloroform extraction) and chromatographic (silica columns, magnetic separation) methods.

    DNA Isolation from Oral Cavity Epithelium

    • Using a swab, epithelial cells are collected from the cheek's inner surface.
    • GeneMATRIX Swab-Extract DNA Purification Kit is used.
    • Buffers (Lyse S, Sol S) and enzyme (Proteinase K) help to break down cells/proteins, releasing DNA.
    • A spin column is used to purify DNA from other cell components.
    • DNA samples are stored at -20°C to prevent degradation.
    • Swabs need to be processed rapidly to avoid DNA degradation.
    • Quantity and purity are confirmed using spectrophotometry.
    • The expected yield is 1–10 µg DNA (10–100 ng/µl).
    • Isolation procedure follows specific steps, including buffer additions, incubation times, centrifugation speeds, and volumes.

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    Related Documents

    Exercise 4: DNA Isolation from Oral Cavity Epithelium - PDF

    Description

    Explore the fundamentals of DNA isolation, including its structure, components, and significance in research. This quiz covers the properties of nucleotides, the double helix formation, and various applications in medical biology and genetic engineering. Test your knowledge on this essential topic in genetics.

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