Blotting and Probing Techniques

“Blotting,” in relation to molecular diagnostics, is a term that refers to the process of detecting the presence and quantity of DNA, RNA, or protein in cells. There are three main types of blotting procedures that those in the field should be familiar with: Southern, Northern, and Western. Three additional blotting procedures are termed Southwestern, Eastern, and Far-Eastern. These are also summarized in the table below.

Southern Blot Steps

1. DNA is isolated and cut with restriction enzymes.
2. The DNA fragments are then analyzed by gel electrophoresis and separated by size (see previous blog post on Separation and Detection).
3. Depurination – Gel is soaked in hydrogen chloride (HCl) to remove the purine bases from the sugar-phosphate backbone. This loosens up larger fragments before denaturation.
4. Denaturation – The DNA is denatured by exposing the gel to sodium hydroxide (NaOH). Denaturation breaks the hydrogen bonds that hold the DNA strands together.
5. Blotting – The denatured DNA is transferred to a solid substrate (nitrocellulose) that helps to facilitate probe binding and signal detection.
6. Pre-hybridization – Prevents non-specific binding of the probe to other sites on the membrane surface.
7. The membrane is exposed to the hybridization probe, usually a single DNA fragment with a specific sequence to the target DNA. The probe DNA is labelled either with radioactivity or fluorescent dyes.
   

Importance of the Membrane

Nitrocellulose and nylon membranes are best for smaller sized single stranded DNA fragments. It is compatible with many types of buffers and transfer systems. These membranes work well with protein and nucleic acids.

 

METHODS OF TRANSFER
Capillary Transfer	Ÿ Utilizes capillary movement of the buffer from a soaked paper to the dry paper Ÿ Denatured DNA moves from the gel to the membrane
Electrophoretic Transfer	Ÿ Electric current moves the DNA from the gel to the membrane
Vacuum Transfer	Ÿ The force from suction moves the DNA from the gel to the membrane

 

Northern Blots

Northern blots are used in the laboratory to look at RNA structure and quantity. It’s a powerful method that can measure levels of gene expression, as well as structural abnormalities in RNA.

• Needs to take place in an RNase-free environment.
• The samples are applied directly to an agarose gel.
• The sample is cut out from the gel, soaked in ammonium acetate to remove the denaturant (denaturant is inhibitory to the binding of RNA to nitrocellulose membranes), and stained with acridine orange or ethidium bromide.

Western Blots

Western blots detect proteins and separates them according to their molecular weight or charge

• Run using a polyacrylamide gel with molecular weight standards / markers.
• Utilizes capillary or electrophoretic transfer methods.
• Membrane must be blocked with a solution to prevent binding of the primary antibody probe to the membrane.

 

TYPES OF PROBES
DNA Probes	Southern Blots	Complementary to the target gene
RNA Probes	Northern Blots	Complementary to the target sequence
Protein Probes	Western Blots	Antibodies bind to the target protein

 

SUMMARY OF HYBRIDIZATION TECHNIQUES
Method	Target	Probe	Purpose
Southern Blot	DNA	Nucleic Acid	·      Gene structure
Northern Blot	RNA	Nucleic Acid	·      RNA transcript structure, processing, and gene expression
Western Blot	Protein	Protein	·      Protein processing and gene expression
Southwestern Blot	Protein	DNA	·      DNA binding proteins and gene regulation
Eastern Blot	Protein	Protein	·      Modification to western blot using enzymatic detection ·      Detection of specific agriculturally important proteins
Far-Eastern Blot	Lipids	None	·      Transfer of HPLC-separated lipids to PVDF membranes for analysis by mass spectrometry

 

-LeAnne Noll, BS, MB(ASCP)^CM is a molecular technologist in Wisconsin and was recognized as one of ASCP’s Top Five from the 40 Under Forty Program in 2015.