ABCs of Molecular Techniques

PCR or Polymerization Chain Reaction, is one of the most used techniques in today's labs. This technique is used to amplify a target DNA from a sample to increase the level of that nucleic information to a level that can be seen by gel electrophoresis or for further applications such as cloning. In this video, you will learn how PCR works, and I will introduce different applications that use PCR for various lab projects.

The most common approach to look at DNA amplification is to use a gel electrophoresis approach. Two primary gels are used to separate DNA fragments based on their sizes: SDS gel or agarose gel. The agarose gel is the most commonly used system to separate DNA. The visualization of the DNA is then accomplished by exposing DNA to chemical products called intercalants. Different intercalants available on the market can be used to visualize DNA under UV (e.g., EtBr, Sybr Green, etc.). In this video, I will explain the principles behind the gel electrophoresis system and how to visualize DNA.

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Genes are expressed into RNAs that eventually will be used to regulate several cellular pathways or to produce new proteins. Modifying these genes can give us a tremendous advantage in impacting different pathways or engineering new proteins with unique features that would not occur naturally. Gene editing has revolutionized many sectors, such as agriculture, pharmaceutical, energy, end many more. Potent tools have been developed to edit any DNA information. Some of these techniques, like the CRISPR-Cas9 method, can target a specific part of a genome and modify the exact base pair that needs to be changed. The number of applications behind gene editing is countless. In this video, I will introduce different techniques that can be used in almost all labs to edit nucleic information. I will also present concrete examples of how gene editing has been used to create new drugs that have saved many lives already.

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When working with genes or RNAs it is often very important to know their exact nucleic information (e.g., base pair). To access that information, different techniques have been developed to read each base pair of DNA and RNA molecules over the years. Such techniques are called sequencing. There are different variations of sequencing methods: Sanger, NGS, and Nanopore. In this video, I will introduce some of these techniques, so you understand how sequencing works and the advantages and inconveniences of each sequencing approach.

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Transgenesis or the art of transforming organisms with nucleic information. Transgenesis has multiple applications, such as improving drug functions, adding new proteins to an organism (e.g., plants, mammalian cells, etc.), and many more. Multiple organisms (e.g., bacteria, plants, cell cultures, insects, etc.) can be transformed with new or modified nucleic information. To transform such a variety of organisms, several protocols have been developed.  These protocols have been designed to introduce DNA or RNA inside the cells so it can be expressed transiently or stably. In this video, I will review different transformation protocols and highlight the advantages and inconveniences of most of these popular transformation techniques. Finally, I will give some examples of transgenesis' applications to engineering new organisms.

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Proteins make everything. They are essential for cell structure, transport, signaling, enzymatic reaction, and more. It is unsurprising that studying protein's structure, function, and regulation is so important. Many tools are at our disposal to study the proteomes of many organisms (e.g., animal and plant cells). After extracting proteins, it is essential to measure their concentration using one of the many techniques discussed in this course, like Bradford, BCA, Lowry, etc. Then many techniques exist to study the exact composition of the proteome of cells. For instance, SDS-PAGE is often the first technique used to separate the content of a mix of proteins.

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In situ RNA hybridization & Immunostaining are two techniques used to detect the presence of transcripts or other cell elements (e.g., protein, lipids). Using nucleic or proteic probes, these two techniques can inform when and where transcripts are expressed in a tissue. It will also tell you where and when the cell's proteins are expressed. You can answer many scientific questions regarding a specific project using these two techniques.

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Many molecular techniques use the power of antibodies for their applications. For instance, ELISA, Western blot, & Immunoprecipitation all use antibodies to detect, purify, & quantify the presence of protein(s) in a sample.

Western blot is often used in labs to detect the presence of a specific protein after separation with an SDS PAGE.

Diagnostic labs routinely use ELISA ( Enzyme Link ImmunoSorbent Assay) to detect the presence of specific antigens in a sample (e.g., blood, urine, etc.). 

Immunoprecipitation is used to precipitate a target protein from a mix of proteins to purify protein(s), so it can be used later for other applications.

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RNA interference or RNAi is a molecular techniques used to temporaly shut down the expression of a gene. To turn off a gene, scientists often create knockout mutants. Even if very efficient, creating KO mutants can be very time and labor-consuming. RNAi can temporally knock down the expression of a specific gene or a group of genes. Unlike KO mutants RNAi can only reduce the expression of a gene but not totally turn it off. However, this technique is easy to set up and can provide powerful data to explore the function of a gene or its regulation.

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RT- PCR & Real-Time PCR are two different PCR techniques. RT-PCR is used to study whether a gene is expressed (i.e., qualitative approach). Real-Time PCR, also known as quantitative PCR (qPCR) or RT-qPCR, is used to quantify the expression of genes (i.e., quantitative approach). Even if both techniques give you some information on the expression of genes, both methods use different principles to do so. Make sure to watch this video to find out how they differ.

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Gene expression consists in studying at the transcriptome level. Different approaches can be used to study the number of copies of each RNA. Many labs use quantitative PCR or Real-Time PCR (RT-qPCR) to determine the absolute or relative expression of genes. RT-qPCR can only study the expression of a few genes at a time. Microarray is another technique that can be used to determine the expression of multiple genes simultaneously. But a new technique called RNA sequencing is used more and more by labs to study the complete transcriptome of a sample. RNA sequencing has many advantages that regular RT-qPCR doesn’t have. In this chapter, we will discuss the principles of such techniques, their benefits, and their inconveniences.

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