Introduction

Techniques for molecular biology have evolved rapidly over the past several decades and are crucial in research for understanding the structure and function of macromolecules, i.e., nucleic acids and proteins, particularly in the fields of medicine and agriculture. Undergraduate laboratory experience is essential for providing students with foundational knowledge and skills to conduct research in the lab. This lab book, Molecular Techniques, is designed to help students with little or no research experience understand the associated underlying principles and applications of molecular techniques, and practice those commonly used in molecular biology research. These labs are suitable for undergraduates in biochemistry, biology, and related sciences who have completed courses in genetics, biochemistry, and cell or molecular biology. The book comprises five project-based modules that focus on the manipulation of DNA, RNA, and proteins. Each module details lab exercises that span two to five weeks. Each contains an overview to introduce the subject and a flowchart of the weekly lab exercises, illustrating how the individual labs fit into the larger research project. Weekly lab exercises begin with objectives, followed by background information related to the techniques used, detailed procedures for the exercise, and conclude with data analysis and discussion questions.

Module 1 focuses on the degenerate PCR cloning of grape nucleotide-binding site (NBS) DNA sequences found in most plant disease resistance (R) genes. Expression and regulation of resistance (R) genes play a crucial role in determining a plant’s susceptibility to pathogens. Cloning and manipulating R genes may enhance plant disease resistance. The cloned NBS sequences can be used as selection markers in breeding programs or for cloning entire R genes to be transferred into susceptible plants, thereby enhancing their disease resistance. After obtaining the recombinant NBS clones, students use restriction mapping, Southern blot analysis, and web-based sequence analysis to validate their identities (Module 2).

Module 3 centers on recombineering of Escherichia coli, both with and without the assistance of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR-Cas9). Recombineering, or so-called recombination-mediated genetic engineering, involves incorporating homologous single-stranded or double-stranded DNA into an organism’s genome. Applications of bacterial recombineering include but are not limited to genome mining for finding new microbial natural products, constructing infectious viral clones, mutating viruses to develop vaccines or vectors for gene therapy, and providing a powerful tool for editing phage genomes to obtain mutants that can potentially kill antibiotic-resistant bacteria. CRISPR-Cas9 is another genome-editing tool with numerous applications, including studying cancers and HIV, treating genetic disorders, and designing new grains to enhance nutritional value. Module 3 compares the editing efficiency of using sense versus antisense DNA templates and determines whether CRISPR-Cas9 enhances the recombineering efficiency of E. coli.

Module 4 examines gene expression with reverse transcription quantitative polymerase chain reaction (RT-qPCR), which measures the expression of the two ribulose bisphosphate carboxylase/oxygenase (RubisCO) small subunit genes. RubisCO is the key enzyme in the dark reaction/Calvin cycle of photosynthesis, a critical factor determining crop yield. Understanding the expression and regulation of RubisCO small subunits may help improve crop production. In this module, RT-qPCR is used to investigate the effect of light on the expression of the two small subunits of RubisCO genes.

Module 5 aims to determine whether bovine serum albumin (BSA) or its homologs are present in the hemolymph of the fruit fly (Drosophila melanogaster) and cow serum, using Western blot analysis, a laboratory technique used to measure protein expression levels and detect specific proteins in a mixture, especially those associated with particular tissues or cell types. For this method, proteins are first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based on molecular weight. Separated proteins are then transferred to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane and subsequently probed with antibodies specific to the target proteins. The results illustrate the sensitivity and specificity of Western blot analysis.

For each module, it may be helpful to review previous student lab results published in the papers listed below.

Module 1: Chang M-M, DiGennaro P, and Macula A. 2009. PCR cloning partial nbs sequences from grape (Vitis aestivalis Michx). Biochem & Mol Biol Educ 37 (6):355-360. doi:10.1002/bmb.20320

Module 2: Chang M-M 2022. Plasmid-to-plasmid Southern blot analysis validates the presence of nbs sequences in cloned plasmids. Biochem & Mol Biol Educ 50 (4): 373-380. doi:10.1002/bmb.21642

Module 3: Chang M-M 2025. An undergraduate laboratory on recombineering and CRISPR-Cas9-assisted gene editing in Escherichia coli. Biochem & Mol Biol Educ 53 (5): 555-562. doi:10.1002/bmb.70002

Module 4: Chang M-M, Li A, Feissner F, and Ahmad T. 2016. An RT-qPCR laboratory exercise demonstrates light-dependent AtRBCS1A and AtRBCS3B mRNA expressions in Arabidopsis thaliana leaves. Biochem & Mol Biol Educ 44 (4): 405- 411. doi:10.1002/bmb.20959.

Module 5: Chang M-M and Lovett J 2011. A laboratory exercise illustrating the sensitivity and specificity of Western blot analysis. Biochem & Mol Biol Educ 39 (4): 291-297. doi:10.1002/bmb.20501