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. (B)</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. (A)</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. (C)</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. (C)</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. (A)</p> Signup and view all the answers

At what wavelength does DNA exhibit maximum absorbance?

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

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

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

What does an A260/A280 ratio of 1.5 suggest?

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

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

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

What is the role of proteinase K in DNA isolation?

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

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

<p>Contamination by proteins. (C)</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 (A)</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' (D)</p> Signup and view all the answers

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

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

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

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

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

<p>Three (D)</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 (D)</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' (D)</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 (B)</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. (D)</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. (A)</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 (A)</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. (D)</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. (D)</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. (B)</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 (D)</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. (B)</p> Signup and view all the answers

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

<p>The upper aqueous layer (A)</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 (A)</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 (C)</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 (D)</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 (B)</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 (D)</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 (A)</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 (D)</p> Signup and view all the answers

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

<p>To degrade proteins (A)</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 (D)</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 (C)</p> Signup and view all the answers

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

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

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

<p>1 to 10 µg (C)</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 (C)</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 (C)</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 (A)</p> Signup and view all the answers

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

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

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

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

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

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

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

<p>Cell disintegration and lysis (C)</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 (B)</p> Signup and view all the answers

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

<p>Purification stage (B)</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 (C)</p> Signup and view all the answers

What is the end goal of DNA isolation procedures?

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

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

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

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

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

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

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

What kind of nucleotides does DNA contain?

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

What is the significance of complementary base pairing in DNA?

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

Which sugar is found in the backbone of DNA?

<p>Deoxyribose (A)</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 (B)</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. (D)</p> Signup and view all the answers

Which wavelength is associated with maximum absorbance for nucleic acids?

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

What does an A260/A280 ratio of 1.5 indicate?

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

How is the concentration of DNA calculated using absorbance measurements?

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

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

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

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

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

Flashcards

What is DNA?

Deoxyribonucleic acid (DNA) is a molecule that contains the genetic instructions for the development and function of living organisms.

What are the building blocks of DNA?

DNA is composed of nucleotides, which are made up of a sugar (deoxyribose), a phosphate group, and one of four nitrogenous bases: adenine (A), guanine (G), cytosine (C), or thymine (T).

How do bases pair in DNA?

Adenine (A) pairs with thymine (T), and guanine (G) pairs with cytosine (C) through hydrogen bonds, creating complementary base pairing.

How are the two DNA strands arranged?

The two strands of DNA run in opposite directions, creating an antiparallel structure. One end of one strand is opposite the beginning of the other strand.

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What makes up the DNA backbone?

The sugar and phosphate groups form the backbone of the DNA molecule, while the nitrogenous bases are located inside the helix.

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What is the shape of DNA?

DNA exists as a double helix, winding around a common axis with the base pairs facing inwards and the sugar-phosphate backbone facing outwards.

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How does the sequence of one DNA strand relate to the other?

The sequence of one DNA strand determines the sequence of the other strand due to complementary base pairing.

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What is DNA replication?

DNA replication involves copying the DNA molecule to create two identical DNA molecules. Each new molecule has one original strand and one newly synthesized strand.

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DNA Isolation

The process of separating and purifying DNA from biological samples like blood, tissues, or cells.

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EDTA (Ethylenediaminetetraacetic Acid)

A chemical that prevents blood from clotting and protects DNA from degradation during isolation.

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Cell Disintegration and Lysis

The stage in DNA isolation where cells are broken down to release DNA into a solution.

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Purification

The process of removing unwanted cellular components like proteins and other molecules from the DNA solution.

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DNA Concentration

The stage where the DNA solution is concentrated to increase its purity and remove low-molecular-weight impurities.

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Genomic DNA

DNA extracted from the nucleus of a cell, containing the complete genetic information of an organism.

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Mitochondrial DNA

DNA extracted from mitochondria, the powerhouses of the cell, involved in cellular respiration.

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Plasmid DNA

DNA found in bacteria and other single-celled organisms, often circular and capable of self-replication.

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A260/A280 Ratio

The ratio of absorbance at 260 nm to absorbance at 280 nm, used to assess the purity of DNA or RNA samples.

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Spectrophotometry

A technique used to measure the absorbance of light at specific wavelengths, enabling the assessment of purity and concentration of DNA and RNA.

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Phenol-Chloroform Extraction

A method of DNA extraction that uses organic solvents like phenol and chloroform to separate DNA from other cellular components.

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Detergent (SDS, Triton-X100)

A substance that dissolves fats and oils, used in DNA isolation to break down cell membranes.

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Proteinase K

An enzyme used in DNA isolation to degrade proteins, facilitating the separation of DNA from other cellular components.

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Silica Column Chromatography

A method of DNA extraction that uses a solid matrix (like silica) to capture DNA molecules.

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Magnetic Separation

A method for isolating DNA that uses magnetic beads coated with DNA-binding molecules.

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Cell Lysis

The process of breaking open cells to release their internal components, including DNA.

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Magnetic Bead Isolation

A method of isolating DNA using small beads that bind to DNA. These beads can be manipulated using a magnet to separate DNA from other cellular components.

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Washing Step

A solution used to remove impurities or unwanted substances from the DNA preparation. Typically involves washing the DNA-containing material several times.

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Elution Buffer

A solution that dissolves the purified DNA from the column or beads. Typically contains Tris-HCl buffer.

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De-salting Buffer

A substance that helps to remove impurities from DNA, such as proteins or lipids.

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Cheek swabbing

A simple and safe method to collect epithelial cells using a swab stick with a cotton tip.

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Activation Buffer (Buffer S)

A specialized buffer used to activate the mini-column in a DNA isolation kit by saturating its membranes.

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Mini-Column

A specialized column used in DNA isolation kits to bind and purify DNA from a mixture of cellular components.

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GeneMATRIX Swab-Extract DNA Purification Kit

A kit that contains all the reagents and tools needed for DNA isolation from a sample, such as a cheek swab.

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Centrifuge

A laboratory tool for separating components in a mixture based on their density by spinning at high speed.

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Lyse S buffer

A buffer solution used to lyse cells by disrupting cell membranes and releasing DNA.

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Sol S buffer

A buffer solution used to precipitate DNA by making it less soluble in water.

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Ethanol (96–100%)

A solution containing ethanol, used to precipitate DNA from the lysate.

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DNA binding spin-column

A specialized column that binds DNA and allows for its separation from other cellular components.

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Wash SX1 buffer

A buffer solution used to wash away impurities from the DNA bound to the spin-column.

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Wash SX2 buffer

A buffer solution used to wash away more impurities from the DNA, ensuring higher purity.

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What is the structure of DNA?

The two strands of DNA run in opposite directions, called antiparallel. One strand's 5' end is opposite the other's 3' end, creating a double helix structure.

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How are nucleotides connected in DNA?

DNA is made of nucleotides, which are joined together by 3',5'-phosphodiester bonds to form a long chain. These bonds connect the phosphate group of one nucleotide to the sugar of the next.

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What is DNA isolation?

The process of isolating and purifying DNA from biological samples, such as blood, tissues, or cells. It involves breaking down the cells, removing unwanted components, and concentrating the DNA.

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What is the A260/A280 ratio?

The ratio of absorbance at 260 nm to absorbance at 280 nm, used to evaluate the purity of DNA samples. A higher ratio generally indicates purer DNA.

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What is spectrophotometry?

A technique that measures the absorbance of light at specific wavelengths to determine the purity and concentration of DNA or RNA. It helps scientists understand the quality of their samples.

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A260/A280 and A230/A260 Ratios

A specific ratio used to assess the purity (A260/A280) or contamination (A230/A260) of a DNA sample.

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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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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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