Blotting Techniques

Blotting techniques involve the separation (via electrophoresis) and transfer of DNA, RNA, or proteins onto a blotting membrane. This separation is generally followed by complexing of the target with a labeled molecule for detection. Southern blotting is used to evaluate for specific DNA sequences and may be used in identification of genetic mutations and in forensics. Northern blotting focuses on RNA sequences and is helpful in assessing gene expression. Western blotting identifies proteins and antibodies and has applications in diagnosing infectious diseases, protein abnormalities (such as prion disease), and autoimmune conditions.  Although these tests have good specificity, they have significant disadvantages owing to their expense, labor-intensiveness, and slow turnaround time.

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Editorial responsibility: Stanley Oiseth, Lindsay Jones, Evelin Maza

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Overview

Definition

Blotting is an investigative technique used to detect and identify macromolecules, such as nucleic acids Nucleic Acids Nucleic acids are polymers of nucleotides, organic molecules composed of a sugar, a phosphate group, and a nitrogenous base. Nucleic acids are responsible for storage, replication, and expression of genetic information. The 2 nucleic acids most commonly seen in eukaryotic cells are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic Acids and proteins. This technique is accomplished by:

  • Separation using electrophoresis
  • Transfer to a membrane
  • Detection with a labeled probe

Types of blotting

The 4 basic types of blotting are

  • Southern
  • Northern
  • Western
  • Eastern (rarely used in clinical setting)

Comparison of blotting techniques

Characteristics of Southern, Northern, and Western blots
Technique Southern blot Northern blot Western blot
Target DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure sequences RNA RNA Ribonucleic acid (RNA), like deoxyribonucleic acid (DNA), is a polymer of nucleotides that is essential to cellular protein synthesis. Unlike DNA, RNA is a single-stranded structure containing the sugar moiety ribose (instead of deoxyribose) and the base uracil (instead of thymine). RNA generally carries out the instructions encoded in the DNA but also executes diverse non-coding functions. RNA Types and Structure sequences Protein
Separation Electrophoresis Electrophoresis Electrophoresis
Blotting method Capillary transfer Capillary transfer Electrophoretic transfer
Probe Oligonucleotides Oligonucleotides Antibodies
Common detection methods
  • X-ray
  • Colorimetry
  • Chemiluminescence
  • X-ray
  • Colorimetry
  • Chemiluminescence
  • Colorimetry
  • Chemiluminescence

Electrophoresis

All blotting techniques use electrophoresis, which involves using an electrical field to separate molecules. 

  • Molecules are separated by: 
    • Size
    • Electrical charge
  • Used extensively in analysis of: 
    • DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure
    • RNA RNA Ribonucleic acid (RNA), like deoxyribonucleic acid (DNA), is a polymer of nucleotides that is essential to cellular protein synthesis. Unlike DNA, RNA is a single-stranded structure containing the sugar moiety ribose (instead of deoxyribose) and the base uracil (instead of thymine). RNA generally carries out the instructions encoded in the DNA but also executes diverse non-coding functions. RNA Types and Structure
    • Proteins
  • Types:
    • Cataphoresis: electrophoresis of cations  
    • Anaphoresis: electrophoresis of anions
  • Technique:
    • Sample is loaded onto a gel.
    • Gel is placed in a buffer solution.
    • An electric field is applied.
    • The electrical current separates the molecules.

Procedure

General blotting technique

Blotting techniques follow a general procedure:

  • The target molecule in a sample is isolated.
  • Electrophoresis separates the molecules.
  • The separated contents are transferred (or blotted) onto a filter or membrane to produce a  “print.”
  • The filter/membrane is then exposed to radiolabeled probes and incubated → probe binds to the target molecule
  • The probe and target molecule create bands that can be visualized with X-ray film.
  • Note: probe and detection methods can also use:
    • Colorimetry
    • Chemiluminescence

Southern blotting

  • Restriction enzymes Enzymes Enzymes are complex protein biocatalysts that accelerate chemical reactions without being consumed by them. Due to the body's constant metabolic needs, the absence of enzymes would make life unsustainable, as reactions would occur too slowly without these molecules. Basics of Enzymes are needed to cleave the DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure sample into fragments prior to electrophoresis
  • Prior to blotting, the separated double-stranded DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure are denatured with alkaline solution → single-stranded DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure fragments
  • Blotting method: capillary transfer of DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure from an electrophoresis gel to a filter
  • Uses labeled oligonucleotide probes that are complementary to the target DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure sequence
  • Incubation allows hybridization of the probe with the target DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure.
Procedure for southern blotting

Procedure for Southern blotting:
A: DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure is cleaved with restriction enzymes Enzymes Enzymes are complex protein biocatalysts that accelerate chemical reactions without being consumed by them. Due to the body's constant metabolic needs, the absence of enzymes would make life unsustainable, as reactions would occur too slowly without these molecules. Basics of Enzymes and separated via electrophoresis.
B: DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure fragments are blotted onto a nitrocellulose filter.
C: The filter is exposed to a solution containing a radiolabeled probe, allowing for hybridization with target DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure sequences.
D: Bands on the filter are then exposed to X-ray film for visualization.

Image by Lecturio.

Northern blotting

  • Blotting method: capillary transfer of RNA RNA Ribonucleic acid (RNA), like deoxyribonucleic acid (DNA), is a polymer of nucleotides that is essential to cellular protein synthesis. Unlike DNA, RNA is a single-stranded structure containing the sugar moiety ribose (instead of deoxyribose) and the base uracil (instead of thymine). RNA generally carries out the instructions encoded in the DNA but also executes diverse non-coding functions. RNA Types and Structure from an electrophoresis gel to a filter
  • Uses labeled oligonucleotide probes that are complementary to the target RNA RNA Ribonucleic acid (RNA), like deoxyribonucleic acid (DNA), is a polymer of nucleotides that is essential to cellular protein synthesis. Unlike DNA, RNA is a single-stranded structure containing the sugar moiety ribose (instead of deoxyribose) and the base uracil (instead of thymine). RNA generally carries out the instructions encoded in the DNA but also executes diverse non-coding functions. RNA Types and Structure sequence
  • Incubation allows hybridization of the probe with the target RNA RNA Ribonucleic acid (RNA), like deoxyribonucleic acid (DNA), is a polymer of nucleotides that is essential to cellular protein synthesis. Unlike DNA, RNA is a single-stranded structure containing the sugar moiety ribose (instead of deoxyribose) and the base uracil (instead of thymine). RNA generally carries out the instructions encoded in the DNA but also executes diverse non-coding functions. RNA Types and Structure.

Western blotting

  • Sometimes, sample proteins need to be denatured before application to an electrophoresis gel.
  • Blotting method: Proteins are electrophoretically transferred (using an electric field) from the gel to the blotting membrane.
  • Uses antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins that are specific to the target protein; this antibody may be:
    • Most common: unlabeled (primary) → if so, a secondary, labeled antibody that can bind to the primary antibody is then used
    • Less common: labeled
  • Incubation allows antibody–antigen complexes to form.

Uses

Southern blot

  • Can detect:
    • Small and large DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure mutations:
      • Deletions
      • Duplications
      • Rearrangements
    • Gene methylation changes
  • Applications:
    • Forensics
    • Identifying new disease-causing mutations
    • Diagnosis of genetic conditions
    • Identification of malignancy-specific genetic markers (e.g., BCR-ABL1 gene fusion in CML CML Chronic myeloid leukemia is a malignant proliferation of the granulocytic cell line characterized by a fairly normal differentiation. The underlying genetic abnormality is the Philadelphia chromosome, an abbreviated chromosome 22, resulting from reciprocal (9;22)(q34;q11) translocation. Chronic Myeloid Leukemia)

Northern blot

Northern blotting can be used to assess RNA RNA Ribonucleic acid (RNA), like deoxyribonucleic acid (DNA), is a polymer of nucleotides that is essential to cellular protein synthesis. Unlike DNA, RNA is a single-stranded structure containing the sugar moiety ribose (instead of deoxyribose) and the base uracil (instead of thymine). RNA generally carries out the instructions encoded in the DNA but also executes diverse non-coding functions. RNA Types and Structure levels and gene expression.

Western blot

Identification of antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins:

  • Diagnosis of infectious diseases, including:
    • HIV 
    • Lyme disease Lyme disease Lyme disease is a tick-borne infection caused by the gram-negative spirochete Borrelia burgdorferi. Lyme disease is transmitted by the black-legged Ixodes tick (known as a deer tick), which is only found in specific geographic regions. Patient presentation can vary depending on the stage of the disease and may include a characteristic erythema migrans rash. Lyme Disease
    • Rickettsia Rickettsia Rickettsiae are a diverse collection of obligate intracellular, gram-negative bacteria that have a tropism for vascular endothelial cells. The vectors for transmission vary by species but include ticks, fleas, mites, and lice. Rickettsia infections
  • Identification of abnormal proteins and antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins, including:
    • Prion disease
    • Muscular dystrophy (dystrophin analysis)
    • Autoantibodies (e.g., anti–glomerular basement membrane (GBM) antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins)

Advantages and Disadvantages

Advantages

  • General: good specificity
  • Southern blot: can detect a wide variety of mutations
  • Western blot:
    • Good sensitivity
    • Can evaluate for several target proteins

General disadvantages

  • Requires a large amount of DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure or RNA RNA Ribonucleic acid (RNA), like deoxyribonucleic acid (DNA), is a polymer of nucleotides that is essential to cellular protein synthesis. Unlike DNA, RNA is a single-stranded structure containing the sugar moiety ribose (instead of deoxyribose) and the base uracil (instead of thymine). RNA generally carries out the instructions encoded in the DNA but also executes diverse non-coding functions. RNA Types and Structure
  • Slow, labor Labor Labor is the normal physiologic process defined as uterine contractions resulting in dilatation and effacement of the cervix, which culminates in expulsion of the fetus and the products of conception. Normal and Abnormal Labor-intensive process
  • Expensive
  • Use of radioactive materials can potentially be hazardous.

References

  1. Kevil, C. G., Walsh, L., Laroux, F. S., Kalogeris, T., Grisham, M. B., Alexander, J. S. (1997). An improved, rapid Northern protocol. Biochem Biophys Res Comm 238:277–279.
  2. Steiling, K., Christenson, S. (2021). Tools for genetics and genomics: gene expression profiling. UpToDate. Retrieved December 29, 2021, from https://www.uptodate.com/contents/tools-for-genetics-and-genomics-gene-expression-profiling
  3. Schrijver, I., Zehnder, J.L. (2021). Tools for genetics and genomics: Cytogenetics and molecular genetics. UpToDate. Retrieved December 29, 2021, from https://www.uptodate.com/contents/tools-for-genetics-and-genomics-cytogenetics-and-molecular-genetics
  4. Gottlieb, G.S. (2019). Clinical manifestations and diagnosis of HIV-2 infection. UpToDate. Retrieved December 29, 2021, from https://www.uptodate.com/contents/clinical-manifestations-and-diagnosis-of-hiv-2-infection
  5. Hu, L. (2021). Diagnosis of Lyme disease. UpToDate. Retrieved December 29, 2021, from https://www.uptodate.com/contents/diagnosis-of-lyme-disease
  6. Mitschka, S., Mayr, C. (2020). Northern blot protocol. Retrieved December 29, 2021, from https://www.protocols.io/view/northern-blot-protocol-bqqymvxw
  7. Gavini, K., Parameshwaran, K. (2021). Western blot. StatPearls. Retrieved December 29, 2021, from https://www.ncbi.nlm.nih.gov/books/NBK542290/
  8. Tomar, M. (2016). Types of blotting. Res Rev J Pharmaceutics Nanotechnol. https://www.rroij.com/open-access/types-of-blotting-.php?aid=79704

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