An in silico DNA cloning experiment for the biochemistry laboratory

Abstract

This laboratory exercise introduces students to concepts in recombinant DNA technology while accommodating a major semester project in protein purification, structure, and function in a biochemistry laboratory for junior- and senior-level undergraduate students. It is also suitable for forensic science courses focused in DNA biology and advanced high school biology classes. Students begin by examining a plasmid map with the goal of identifying which restriction enzymes may be used to clone a piece of foreign DNA containing a gene of interest into the vector. From the National Center for Biotechnology Initiative website, students are instructed to retrieve a protein sequence and use Expasy's Reverse Translate program to reverse translate the protein to cDNA. Students then use Integrated DNA Technologies' OligoAnalyzer to predict the complementary DNA strand and obtain DNA recognition sequences for the desired restriction enzymes from New England Biolabs' website. Students add the appropriate DNA restriction sequences to the double-stranded foreign DNA for cloning into the plasmid and infecting Escherichia coli cells. Students are introduced to computational biology tools, molecular biology terminology and the process of DNA cloning in this valuable single session, in silico experiment. This project develops students' understanding of the cloning process as a whole and contrasts with other laboratory and internship experiences in which the students may be involved in only a piece of the cloning process/techniques. Students interested in pursuing postgraduate study and research or employment in an academic biochemistry or molecular biology laboratory or industry will benefit most from this experience.

Understanding the Use of Real Time Reverse Transcriptase-Polymerase Chain Reaction (rRT-PCR) For Covid-19 Diagnosis

“…Primers are designed using in silico primer designing algorithms such as Primer3 (http://primer3.wi.mit.edu) [26], and NCBI Primer blast [27]. The designed primers should also be optimized in silico using platforms at idtdna (https://sg.idtdna.com/ pages/tools/oligoanalyzer) and genomeucsc.edu (https:// genome.ucsc.edu/cgi-bin/hgPcr) [28] before their wet laboratory optimization. Generally, both the preformulated IVDs kits produced by different manufacturers for COVID-19 case confirmation (Table 1) and in-house rRT-PCR protocols recommended by CDC target 1-3 genes within the SARS-CoV-2 genome (Table 1, Figure 2).…”

Curriculum and course materials for a forensic DNA biology course

“…cannabis) DNA typing and analysis, or RNA extraction and tissue differentiation . An in silico DNA cloning laboratory is useful to expose students to principles of modern DNA manipulation as covered in some of the introductory forensic textbooks without the use of expensive reagents and can be used to review DNA biology earlier in the course.…”

The Zebrafish Neurophenome Database (ZND): A Dynamic Open-Access Resource for Zebrafish Neurophenotypic Data

“…[26][27][28][29][30][31][32] Collecting massive amounts of data in publicly available online locations has produced multiple scientific databases that make scientific information available to more scientists in more places. [33][34][35][36][37][38][39][40][41][42][43][44][45] This ''database revolution'' 47,48 has allowed researchers to apply in silico approaches (in addition to in vivo and in vitro experiments [49][50][51][52][53][54] ) to analyze large amounts of data rapidly, as well as to deconstruct and re-integrate highly specific aspects of their data, leading to a greater collective understanding of neurobiological phenomena.…”