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
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
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
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.