Isolation and purification of DNA.

Isolation and purification of DNA involve extracting DNA from cells and removing contaminants to obtain a pure sample for downstream applications. Here's an overview of the methods commonly used for DNA isolation and purification:

1. Basic Steps of DNA Isolation

  1. Cell Lysis: Breaking open the cells to release their contents, including DNA.
  2. DNA Separation: Separating DNA from other cellular components like proteins, lipids, and RNA.
  3. DNA Purification: Removing contaminants to obtain pure DNA.
  4. DNA Precipitation: Concentrating DNA by precipitating it out of the solution.

2. Common Methods for DNA Isolation

A. Organic Extraction (Phenol-Chloroform Method)

  • Procedure:
    1. Cell Lysis: Cells are lysed using a detergent and proteinase K to break down proteins and membranes.
    2. Phenol-Chloroform Extraction: An equal volume of phenol-chloroform is added to the lysate. The mixture is centrifuged to separate into two phases: an aqueous phase (containing DNA) and an organic phase (containing proteins and lipids).
    3. DNA Precipitation: The aqueous phase is collected, and DNA is precipitated by adding cold ethanol or isopropanol.
    4. DNA Washing: The DNA pellet is washed with ethanol to remove any residual contaminants.
    5. DNA Resuspension: The DNA is dissolved in a suitable buffer (e.g., TE buffer).
  • Advantages: High yield and purity.
  • Disadvantages: Involves toxic chemicals like phenol and chloroform.

B. Salting Out Method

  • Procedure:
    1. Cell Lysis: Cells are lysed using a detergent.
    2. Protein Precipitation: High salt concentration (e.g., sodium chloride) is added to precipitate proteins, which are then removed by centrifugation.
    3. DNA Precipitation: DNA is precipitated by adding ethanol or isopropanol.
    4. DNA Washing and Resuspension: The DNA pellet is washed with ethanol and dissolved in a buffer.
  • Advantages: Simple and avoids toxic chemicals.
  • Disadvantages: May not achieve the same purity level as organic extraction.

C. Silica Column-Based Kits

  • Procedure:
    1. Cell Lysis: Cells are lysed using a buffer containing a detergent.
    2. Binding to Silica: The lysate is applied to a silica column, where DNA binds to the silica membrane in the presence of chaotropic salts.
    3. Washing: The column is washed with ethanol-containing buffers to remove contaminants.
    4. Elution: Pure DNA is eluted from the column using a low-salt buffer or water.
  • Advantages: Quick, convenient, and produces high-purity DNA.
  • Disadvantages: Can be more expensive than other methods.

D. Magnetic Bead-Based Kits

  • Procedure:
    1. Cell Lysis: Cells are lysed using a lysis buffer.
    2. Binding to Magnetic Beads: DNA binds to magnetic beads coated with a DNA-binding substance.
    3. Washing: Beads are washed to remove contaminants using a magnetic separator.
    4. Elution: DNA is eluted from the beads.
  • Advantages: Automation-friendly and suitable for high-throughput applications.
  • Disadvantages: Costly compared to manual methods.

E. Alkaline Lysis (Plasmid DNA Isolation)

  • Procedure:
    1. Cell Lysis: Bacterial cells are lysed using an alkaline lysis solution.
    2. Neutralization: The lysate is neutralized, causing proteins and chromosomal DNA to precipitate while plasmid DNA remains in solution.
    3. Centrifugation: The mixture is centrifuged to remove precipitates.
    4. DNA Precipitation: Plasmid DNA is precipitated using ethanol or isopropanol.
  • Advantages: Efficient for plasmid DNA isolation.
  • Disadvantages: Not suitable for genomic DNA isolation.

3. DNA Purity and Concentration Assessment

  • Spectrophotometry: The purity and concentration of DNA are assessed using a spectrophotometer by measuring absorbance at 260 nm (DNA) and 280 nm (protein). A pure DNA sample has an A260/A280 ratio of ~1.8.
  • Agarose Gel Electrophoresis: Used to check the integrity of the isolated DNA by visualizing it on a gel.

4. Key Considerations

  • Sample Type: The choice of method may vary depending on the sample type (e.g., blood, tissue, plant).
  • Downstream Application: The required purity and yield depend on the intended application (e.g., PCR, cloning, sequencing).

Comments

Post a Comment

Popular posts from this blog

Watson -Crick Model of DNA structure

Image
The Watson-Crick model of DNA, proposed by James Watson and Francis Crick in 1953, is one of the most significant scientific discoveries of the 20th century. This model describes the double-helix structure of DNA, which is the molecule responsible for carrying genetic information in living organisms. Key Features of the Watson-Crick Model: Double Helix Structure : DNA is composed of two long strands that coil around each other to form a double helix. The strands are antiparallel, meaning they run in opposite directions (5' to 3' and 3' to 5'). Backbone Composition : Each strand of DNA consists of a sugar-phosphate backbone. The backbone is made up of alternating deoxyribose sugars and phosphate groups. The sugar in DNA is deoxyribose, a five-carbon sugar. Base Pairing : The two strands of the DNA helix are held together by hydrogen bonds between pairs of nitrogenous bases. There are four types of bases in DNA: Adenine (A) Thymine (T) Cytosine (C) Guanine (G) Adenine pai...
Read more

A, B and Z–DNA, Cruciform - structure in DNA

DNA can adopt several different conformations beyond the classic B-DNA form. The most well-known are A-DNA, B-DNA, and Z-DNA, each with distinct structural characteristics. Additionally, certain sequences in DNA can form a cruciform structure under specific conditions. A-DNA Structure : A-DNA is a right-handed double helix like B-DNA, but it is more compact. The helix is shorter and wider, with 11 base pairs per turn, compared to B-DNA's 10.5. Backbone : The sugar-phosphate backbone is closer to the helical axis, making the grooves less pronounced. The major groove is deep and narrow, while the minor groove is shallow and broad. Helical Diameter : Approximately 23 Å. Conditions : A-DNA typically forms under conditions of low humidity or dehydration. It is not as common in living cells but can occur in DNA-RNA hybrids and in regions where DNA is bound to certain proteins. Function : Although not prevalent in cells, A-DNA may play a role in DNA-protein interactions and the regulation...
Read more

Secondary and tertiary structure of RNA

 RNA molecules are versatile and can form complex structures that are crucial for their functions. The secondary and tertiary structures of RNA are essential to understanding how RNA molecules perform their roles in cells. 1. Secondary Structure of RNA: Definition : The secondary structure of RNA refers to the local base-pairing interactions within an RNA molecule, which create structures like helices, loops, and bulges. Key Elements : Hairpins/Stem-loops : These are formed when a sequence of nucleotides pairs with a complementary sequence downstream, leaving a loop of unpaired bases at the top. Bulges : Occur when there are unpaired nucleotides on one strand within a helix. Internal Loops : Regions where both strands of the helix have unpaired bases. Multibranched Junctions : Points where three or more helices converge. Importance : RNA secondary structures are stabilized by hydrogen bonding between complementary bases (A-U and G-C) and are important for the RNA’s stability and fu...
Read more