DNA Isolation Techniques and Applications 3

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Questions and Answers

What is the main objective of isolating genomic DNA?

To identify the sequence of DNA.

To create copies of DNA.

To study the structure of DNA.

To obtain high molecular weight DNA purified from proteins and enzyme inhibitors. (correct)

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

Bone marrow (correct)

Epithelial cells

Peripheral blood

Hair follicles

What is the purpose of EDTA in blood collection tubes for DNA isolation?

To prevent blood clotting and inhibit the activity of deoxyribonucleases. (correct)

To activate enzymes responsible for DNA replication.

To preserve the integrity of red blood cells.

To enhance blood clotting and promote DNA degradation.

What is the primary consideration in choosing a DNA isolation method?

The type of nucleic acid to be isolated. (D) Signup and view all the answers

Which of the following is NOT a potential application of isolated genomic DNA?

Diagnosis of bacterial infections (A) Signup and view all the answers

Which of the following methods is NOT a method for purifying DNA?

Magnetic separation (B) Signup and view all the answers

What is the purpose of using chaotropic salts in DNA isolation by the column method?

To increase the binding of DNA to the ion-exchange membrane (A) Signup and view all the answers

Which of the following reagents is commonly used for DNA precipitation in organic methods?

Ethanol (D) Signup and view all the answers

In the salt method of DNA isolation, what is the purpose of adding NaCl?

To precipitate proteins (D) Signup and view all the answers

Which of the following is NOT a common contaminant found in DNA and RNA preparations?

Lysis buffer (D) Signup and view all the answers

What is the purpose of the washing step in the column method of DNA isolation?

To remove impurities from the DNA (A) Signup and view all the answers

Which of the following techniques is NOT used to determine the purity and concentration of DNA?

Magnetic separation (D) Signup and view all the answers

Why is it important to pre-dilute DNA samples before spectrophotometric measurement?

To increase the accuracy of the measurement (A) Signup and view all the answers

Which of the following techniques can be used to homogenize soft animal tissues?

Homogenization (A) Signup and view all the answers

What is the primary purpose of protein lysis (denaturation) during DNA isolation?

To inactivate nucleases and prevent DNA degradation (A) Signup and view all the answers

Which of these methods is a common technique for isolating DNA from bacterial cells and cell cultures?

Chemical lysis using chaotropic salts (D) Signup and view all the answers

Which of the following is NOT a commonly used material for diagnostic studies?

Urine (B) Signup and view all the answers

What is the ideal storage condition for biological material that cannot undergo DNA isolation within 48 hours?

-80°C (D) Signup and view all the answers

How might a sample be protected during transportation?

Transport in a padded container (D) Signup and view all the answers

What is a crucial step to ensure proper DNA isolation?

Proper labeling and sealing of the sample container (A) Signup and view all the answers

Why is it essential to inactivate nucleases during the DNA isolation process?

Nucleases can degrade DNA, compromising its quality (D) Signup and view all the answers

Study Notes

Isolation of Genomic DNA

The goal is to isolate high-molecular-weight DNA, free of proteins and enzyme inhibitors.
This purified DNA is crucial for subsequent DNA-related work.
Applications include genetic disease detection, cancer predisposition assessment, next-generation drug production, cell therapy, and genetic engineering.

DNA Structure

DNA is a double helix.
The double helix is composed of a sugar-phosphate backbone.
Nucleotides, containing bases (adenine, thymine, cytosine, and guanine), form the "rungs" of the ladder.
Hydrogen bonds connect paired bases (A with T, and C with G).

Choosing a DNA Isolation Method

The best method depends on the type of nucleic acid needed (genomic, mitochondrial, plasmid).
The source material's origin (plant, animal, bacterial, viral).
The material type (tissues, organs, cell cultures).
The expected results (purity, quality, time).
The intended purpose (PCR, cloning, etc.).

Materials for Isolation (Human)

Peripheral blood is a common source, isolated using EDTA tubes.
EDTA prevents blood clotting and inhibits deoxyribonucleases (DNases).
Heparin-containing tubes can negatively impact subsequent stages.
Other sources include epithelial cells, fibroblast cultures, amniotic fluid cells (AFC), chorionic villus sampling (CVS), and hair follicles.
Less common materials include blood spots, semen, tissue fragments, and bone marrow.

Stages of DNA Isolation

Collection of the biological material sample.
Transport of the collected material.
Homogenization of the collected material.
Protein lysis (denaturation).
Purification of DNA from lysate remnants.
Dissolving DNA.
Qualitative and quantitative evaluation of the isolated DNA.

Transport

Proper labeling is essential.
Secure sealing of tubes or containers is necessary.
Protection from damage and contamination is crucial.
Protection from temperature changes during transport is needed.

Storage

Immediately begin DNA isolation after sample collection.
Tissues can be stored at 4°C for several days without significant DNA degradation.
If isolation isn't possible within 48 hours of collection, freeze at -20°C or -80°C.
Longer storage reduces both yield and DNA quality.

Homogenization

The method depends on material origin.
Soft animal tissues: homogenization.
Cell suspensions and biological fluids: sonication (ultrasound) or chemical lysis.
Plant cells: mechanical grinding.
Bacterial cells and cell cultures: chemical lysis.

Cell Lysis

Disrupts cell membranes to release DNA/RNA.
Denatures proteins and inactivates nucleases.
Methods: Enzymatic (proteinase K, collagenase, trypsin, chitinase), Chemical (chaotropic salts like guanidine isothiocyanate, detergents like CTAB, SDS, Triton X-100), Physical (heating, freezing, osmotic shock), and Mechanical (glass beads, aluminum oxides).

DNA Isolation Methods

Solvent-based: Phenol-chloroform extraction, protein precipitation.
Chromatographic: Binding DNA to carrier material (silica columns), magnetic separation.

Purification of DNA (organic method)

Centrifugation to separate DNA from associated compounds.
Use of a saturated phenol solution buffer, chloroform with isoamyl alcohol, mixture of phenol with chloroform and isoamyl alcohol (25:24:1).

Purification of DNA (salt method)

Lysis, followed by centrifugation after adding sodium chloride.
Precipitation process.

DNA Isolation by Column Method

DNA binds to ion-exchange membranes, removing contaminants.
The column is washed to remove impurities.
DNA is eluted (removed from the membrane) using a low-salt buffer.

DNA Isolation by Magnetic Method

DNA binds to paramagnetic beads.
Removal of contaminants using washing steps.
DNA is eluted into solution.

Quantification and Quality Assessment

Crucial step for genetic material work.
Contaminants (phenol, EDTA, heparin) can negatively impact analytical techniques (especially PCR).
Methods for measuring DNA concentration: spectrophotometry, agarose gel electrophoresis.

Spectrophotometric Measurement

Dilute DNA/RNA samples with water or TE buffer.
Measure absorbance at specific wavelengths:
- 260 nm (DNA/RNA absorb strongly).
- 280 nm (proteins absorb).
Ratio of absorbance at 260nm/280nm (A260/A280) indicates purity, typical ratios are:
- dsDNA ~1.8.
- RNA ~2.0.
- Values below 1.8 suggest protein contamination, 1.5 ≈ 50% contamination.

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