RESEARCH TECHNIQUES MADE SIMPLE: Polymerase Chain Reaction (PCR) Q&A

The questions and answers below relate to the Research Techniques Made Simple article entitled “Polymerase Chain Reaction” published in the March 2013 issue of the Journal of Investigative Dermatology

 

Questions

 

1. Qualitative PCR and quantitative PCR provide information on ____________ and ________ respectively.

A. presence/absence of specific DNA product; how much of a specific DNA product is present

B. how much of a specific DNA product is present; presence/absence of DNA product

C. RNA; DNA

D. gene byproducts; RNA

 

Answer:  A. presence/absence of specific DNA product; how much of a specific DNA product is present. Qualitative PCR is used to detect the presence or absence of a specific DNA product.  Qualitative PCR is a good technique to use when PCR is performed for cloning purposes or for identification of a pathogen. On the other hand, quantitative PCR provides more information beyond just mere detection of DNA.  It is able to indicate how much of a specific DNA or gene is present in the sample.

 

 

2. The most widely used method of analysis of the PCR product is:

A. agarose gel electrophoresis

B. western blot

C. ELISA

D. FISH

 

Answer:  A. agarose gel electrophoresis. The most widely used method of analysis of the PCR product is via the use of the simple agarose gel electorophoresis.  The agarose gel electrophoresis is the easiest and most common method of visualizing and analyzing the PCR product.  It allows the determination of the presence and the size of the PCR product.   A predetermined set of DNA products with known sizes are ran on the gel as molecular markers to help determine the size of the product. The two other methods for visualizing the PCR products are staining of the amplified DNA product using a chemical dye such as ethidium bromide, which intercalates between the two strands of the duplex or labeling the PCR primers or nucleotides with fluorescent dyes (fluorophores) prior to PCR amplification.

 

 

3. A major advantage of using PCR as compared to other molecular biology techniques is:

A. low risk of contamination

B. rapidity

C. low sensitivity

D. low specificity

 

Answer:  B. rapidity. PCR allows the creation of billions of copies of a specific DNA fragment or gene, which allows for detection and identification of gene sequences using visual techniques based on size and charge. It is a highly sensitive technique and can allow results within a shorter time frame than most molecular techniques.

 

 

4. The PCR process contains these three steps:

A. denaturation, transcription, annealing

B. annealing, denaturation, transcription

C. denaturation, annealing, transcription

D. transcription, annealing, denaturation

 

Answer:  C. denaturation, annealing, transcription. The PCR process can be divided into three main steps. The denaturation process allows the solution to be heated above the melting point of the two complementary strands of the template DNA, which allows the strands to separate. Then the annealing process allows the primers to bind to the specific DNA segment. The annealing between the primers and the template DNA occurs only if they are complementary in sequence. The temperature is raised again at which time the DNA polymerase is able to extend the primers by adding nucleotides to the developing DNA strand, known as transcription. With each repeat of these three steps, the number of copied DNA molecules is doubled.

Why Massage Feels So Good

New Uses for Old Drugs

Julie S. Green, MD, PhD, University of Colorado Denver
Lowell A. Goldsmith, MD, MPH, University of North Carolina

 

Modern drug design and development is often mechanistically and rationally based.  One such example is the TNF-α inhibitor class of agents, which were developed based on evidence that levels of TNF-α and IL-1 are increased in autoimmune inflammatory diseases of the joint and the gastrointestinal tract (Brynskov et al., 1992; Saxne et al., 1988; Tetta et al., 1990).  Improvement of cutaneous psoriatic lesions during treatment of psoriatic arthritis led to approval of several TNF-α inhibitors to treat psoriasis (Mease et al., 2000), therapies that have revolutionized the approach to treating this physically and emotionally debilitating disease.

Another method by which treatments for diseases are developed involves the expansion over time of indications for which a particular drug is used after its initial FDA approval for a  limited set of indications.   After FDA approval and release of a drug onto the market, clinical experience is gathered from an often much larger patient population than was employed during clinical trials; experience accumulated over years of usage often informs clinicians about various side effects of the medication, both adverse and serendipitous.  Such accumulated experiences, if significant, may grant more expeditious approval of a new indication for a drug.  There are a number of examples of agents currently used for dermatologic indications that were initially developed for other purposes.

One noteworthy such example is thalidomide, developed in the 1950s as a hyponotic and an anticonvulsant.  This drug was licensed in 1956 as an over-the-counter sleeping aid in Europe and in other parts of the world.  By 1959, an increased incidence of phocomelia, an otherwise rare condition characterized by shortening of the limbs, was being reported, but its association with maternal thalidomide use was not suggested until 1961 (Mellin and Katzenstein, 1962).  By the time thalidomide was removed from the market, over 10,000 children had been born with thalidomide embryopathy.  Though the drug could have been permanently removed from the market, it was found to be an effective treatment for erythema nodosum leprosum (Sheskin, 1965), which remains the only FDA-approved indication for thalidomide.  A rigorous screening and monitoring program (System for Thalidomide Education and Prescribing Safety [S.T.E.P.S.])  administered by the manufacturer of thalidomide ensures that patients and physicians understand the potential risks of the medication and that no patient who might become pregnant gains access to the drug.

Now, Kim et al (2013) describe in the Journal of Investigative Dermatology the use of ketotifen, an agent currently FDA-approved for the prevention of asthma exacerbations and for treating allergic eye disease, as a potential treatment to reduce the effects of UV damage on chronically sun-exposed skin.   The authors report that ketotifen attenuates mast cell recruitment and matrix metalloproteinase (MMP) activation and reduces UV-induced wrinkling in hairless mice.  These findings apply established knowledge of both ketotifen’s mechanism of action and the  processes that underlie UV-induced photoaging to investigate a potential novel treatment strategy for this phenomenon.  As understanding of the pathophysiological basis of disease increases, novel uses for existing medications with well-elucidated physiologic effects and development of new therapeutics targeted to specific pathologic processes are both likely to remain important strategies for developing new treatments for various dermatologic diseases.

 

REFERENCES

Brynskov J, Tvede N, Andersen C, Vilien M (1992) Increased production of interleukin-1alpha, interleukin-2 and soluble interleukin-2 receptor in endoscopic mucosal biopsy specimens with active inflammatory bowel disease. Gut 33:55-8.

Kim M-S, Lee DH, Lee C-W, et al.  Mast Cell Stabilizer, Ketotifen, Prevents UV_Induced Wrinkle Formation.  J Invest Dermatol doi:  10.1038/jid.2012.424

Mease P, Goffe B, Metz J, VanderStoep A, Finck B, Burge D (2000) Etanercept in the treatment of psoriatic arthritis and psoriasis. Lancet 356:385-90.

Mellin GW, Katzenstein M (1962) The Saga of Thalidomide — Neuropathy to Embryopathy, with Case Reports of Congenital Anomalies. New Engl J Med 267:1238-44.

Saxne T, Palladino M, Heinegård D, Talal N, Wollheim F (1988) Detection of tumor necrosis factor alpha but not tumor necrosis factor beta in rheumatoid arthritis synovial fluid and serum. Arthritis Rheum 31:1041-5.

Sheskin J (1965) Thalidomide in the treatment of lepra reactions. N Engl J Med 6:303-6.

Tetta C, Camussi G, Modena V, Di Vittorio C, Baglioni C (1990) Tumour necrosis factor in serum and synovial fluid of patients with active and severe rheumatoid arthritis. Ann Rheum Dis 49:665-7.