Undergraduate virology exercises demonstrate conventional and real‐time PCR using commercially available HIV primers and noninfectious target

Abstract

It is an extraordinary challenge to offer an undergraduate laboratory course in virology that teaches hands-on, relevant molecular biology techniques using nonpathogenic models of human virus detection. To our knowledge, there exists no inexpensive kits or reagent sets that are appropriate for demonstrating real-time PCR (RT-PCR) in an undergraduate laboratory course in virology. Here we describe simple procedures for student exercises that demonstrate the PCR detection of an HIV target nucleic acid. Our procedures combine a commercially available kit for conventional PCR with a modification for RT-PCR using the same reagents in the kit, making it possible for an instructor with access to a LightCycler 1 instrument to implement a relevant student exercise on RT-PCR detection of HIV nucleic acid targets. This combination of techniques is useful for demonstrating and comparing conventional PCR amplification and detection with agarose gel electrophoresis, with real-time PCR over a series of three laboratory periods. The series of laboratory periods also is used to provide the foundation for teaching the concept of PCR primer design, optimization of PCR detection systems, and introduction to nucleic acid queries using NCBI-BLAST to find and identify primers, amplicons, and other potential amplification targets within the HIV viral genome. The techniques were successfully implemented at the Biology 364 undergraduate virology course at the University of Scranton during the Fall 2008 semester. The techniques are particularly targeted to students who intend to pursue either postgraduate technical employment or graduate studies in the molecular life sciences.

Virology in the Classroom: Current Approaches and Challenges to Undergraduate- and Graduate-Level Virology Education

“…Two published papers report on application of PCR, real-time PCR, and BLAST analysis to a virological situation. In one, using three lab periods, students use BLAST to design PCR primers and to identify amplification targets, work on optimization of PCR, and use PCR and real-time PCR to amplify a portion of the HIV-1 env gene from a provided DNA template (20). In a second, using as few as two lab sessions (or as many as four), students obtain nasal lavage samples, isolate RNA and synthesize complementary DNA, perform PCR and quantitative PCR, and analyze products using gel electrophoresis and sequencing/BLAST.…”

Novel primary literature‐based alternative to comprehensive final examination for undergraduate virology course

“…Until 2010, there was an optional 2.0 credit laboratory component to the virology course. The present author described the use a non‐infectious technique that uses commercially available PCR primers and template as a model for HIV diagnosis for the concurrent laboratory component of the course 1 …”

“…The present author described the use a non-infectious technique that uses commercially available PCR primers and template as a model for HIV diagnosis for the concurrent laboratory component of the course. 1 The lecture component of the course stressed molecular virology and clinical applications: molecular and serological diagnostic protocols, vaccination strategies, and antiviral chemotherapy of viral diseases affecting humans.…”

RFLP analysis and allelic discrimination with real‐time PCR using the human lactase persistence trait

“…Several excellent laboratory exercises are available to teach undergraduate students about aspects of traditional PCR and in some cases, post‐PCR analysis, including primer design [2, 3]; DNA isolation, PCR, and electrophoresis [4–7]; and population analysis of allelic frequency using the Hardy–Weinberg equations [8]. Some of these exercises use human DNA [5, 8, 9], whereas others use DNA from fruit flies [6], mushrooms [7], or viruses [10]. One 4‐week laboratory investigation of the “God Gene,” in which PCR and DNA sequencing of polymorphic sites is used in a nonmajors course is also available [9].…”

“…One 4‐week laboratory investigation of the “God Gene,” in which PCR and DNA sequencing of polymorphic sites is used in a nonmajors course is also available [9]. Currently, real‐time PCR has been adapted to the undergraduate laboratory in only one application of which we are aware [10].…”