Month: May 2016

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

 

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-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.

 

 

 

Microbiology Case Study: A 52 Year Old Woman’s Routine Colonoscopy Results

A 52 year old woman with no significant past medical history presented for a routine colonoscopy screening. During the colonoscopy, the mucosa of the colon was abnormal with a vascular pattern that was distorted. There were whitish punctate lesions, particularly in the right colon and a biopsy was taken.

H&E stain of colon biopsy
H&E stain of colon biopsy
H&E stain of colon biopsy
H&E stain of colon biopsy

Based on the microscopic morphology, a diagnosis of schistosomiasis was made. Evaluation of the morphology reavealed lateral spines which is consistent with the diagnosis of Schistosoma mansoni.

Schistosomiasis is a disease caused by infection with parasitic blood flukes. The parasites that cause schistosomiasis live in certain types of freshwater snails. The infectious form of the parasite is known as cercariae. Individuals can become infected when skin comes in contact with contaminated water and is penetrated by cercariae.

Five schistosome species can cause infection in humans:

  • Schistosoma mansoni (Africa and South America)
  • S. japonicum(East Asia)
  • S. haematobium(Africa and Middle East).
  • S. mekongi(Laos, Cambodia)
  • S. intercalatum(West and Central Africa);

The adult worms travel against portal blood flow to the mesenteric venules of the colon. The male schistosome forms a groove for the female in which mating occurs, and in 1-3 months, the females deposit eggs in the small venules of the mesenteric or perivesical systems. The eggs of S. mansoni and S. japonicum move toward the lumen of the intestine while the eggs of S. haematobium move toward the bladder and ureters. They leave the body in feces or urine. Adult worms have an average lifespan of 5-7 years but have been known to survive up to 30 years

S. mansoni and S. japonicumgenerally cause intestinal tract disease and S. haematobiumcauses genitourinary tract disease. More than 200 million people have been infected, leading to approximately 200,000 deaths per year.

Most people infected do not develop symptoms, however infection can lead to swimmer’s itch, acute schistosomiasis syndrome (sudden onset of fever, urticaria and angioedema, chills, myalgias, arthralgias, dry cough, diarrhea, abdominal pain, and headache), and intestinal schistosomiasis (abdominal pain, poor appetite, and diarrhea).

The acute phase of infection is treated with corticosteroids. Praziquantel should be started after acute symptoms have resolved and should be given with corticosteroids.

 

-Mustafa Mohammed, MD is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.