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Base Pairing: Pairing of purine and pyrimidine bases by Hydrogen bonding in double-stranded DNA or RNA.

What are Nucleic Acids?

JoVE 10684

Nucleic acids are long chains of nucleotides linked together by phosphodiester bonds. There are two types of nucleic acids: deoxyribonucleic acid, or DNA, and ribonucleic acid, or RNA. Nucleotides in both DNA and RNA are made up of a sugar, a nitrogen base, and a phosphate molecule.

A cell’s hereditary material is comprised of nucleic acids, which enable living organisms to pass on genetic information from one generation to next. There are two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA and RNA differ very slightly in their chemical composition, yet play entirely different biological roles. Chemically, nucleic acids are polynucleotides—chains of nucleotides. A nucleotide is composed of three components: a pentose sugar, a nitrogen base, and a phosphate group. The sugar and the base together form a nucleoside. Hence, a nucleotide is sometimes referred to as a nucleoside monophosphate. Each of the three components of a nucleotide plays a key role in the overall assembly of nucleic acids. As the name suggests, a pentose sugar has five carbon atoms, which are labeled 1o, 2o, 3o, 4o, and 5o. The pentose sugar in RNA is ribose, meaning the 2o carbon carries a hydroxyl group. The sugar in DNA is deoxyribose, meaning the 2o

 Core: Macromolecules

RNA Structure

JoVE 10799

The basic structure of RNA consists of a five-carbon sugar and one of four nitrogenous bases. Although most RNA is single-stranded, it can form complex secondary and tertiary structures. Such structures play essential roles in the regulation of transcription and translation.

There are three main types of ribonucleic acid (RNA): messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). All three RNA types consist of a single-stranded chain of nucleotides. Each nucleotide is composed of the five-carbon sugar ribose. The carbon molecules of ribose are numbered one through five. Carbon number five carries a phosphate group and carbon number one a nitrogenous base. There are four nitrogenous bases in RNA—adenine (A), guanine (G), cytosine (C), and uracil (U). Uracil is the only base in RNA that is not present in DNA, which uses thymine (T) instead. During transcription, RNA is synthesized from a DNA template based on complementary binding of the new RNA bases to the DNA bases; A binds to T, G binds to C, C binds to G, and U binds to A. Like DNA, adjacent nucleotides in RNA are linked together through phosphodiester bonds. These bonds form between the phosphate group of one nucleotide and a hydroxyl (–OH) group on the ribose of the adjacent nucleotide. This structure lends RNA its directionality—that is, the two ends

 Core: Gene Expression

MicroRNAs

JoVE 10801

MicroRNA (miRNA) are short, regulatory RNA transcribed from introns—non-coding regions of a gene—or intergenic regions—stretches of DNA present between genes. Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA ends have been methylated to prevent degradation, it is exported from the nucleus into the cytoplasm. In the cytoplasm, another endonuclease enzyme, called Dicer, cuts the pre-miRNA into a 21-24 nucleotide-long miRNA duplex. Then, Dicer cleaves one strand of the duplex, releasing a single strand of mature miRNA. The mature miRNA is loaded into a protein complex called RNA-induced silencing complex (RISC), which the miRNA then guides to the complementary region of its target mRNA. The extent of complementary base-pairing between miRNA and 3’ untranslated region of target mRNA determines the gene silencing mechanism. Extensive or near-perfect complementarity causes degradation of mRNA, whereas limited base-pairing inhibits translation. While silencing via mRNA degradation is irreversible, translation inhibition is reversible since stable mRNA can

 Core: Gene Expression

PCR

JoVE 10819

The polymerase chain reaction, or PCR, is a widely used technique for copying segments of DNA. Due to exponential amplification, PCR can produce millions or billions of DNA copies within just a few hours. In a PCR reaction, a heat-resistant DNA polymerase enzyme amplifies the original DNA through a series of temperature changes inside an automated machine called a thermocycler.

Kary Mullis developed PCR in 1983, for which he was awarded the 1993 Nobel Prize in Chemistry. Being a relatively fast, inexpensive, and precise way of copying a DNA sequence, PCR became an invaluable tool for numerous applications, including molecular cloning, gene mutagenesis, pathogen detection, gene expression analysis, DNA quantitation and sequencing, and genetic disease diagnosis. PCR mimics the natural DNA replication process that occurs in cells. The reaction mixture includes a template DNA sequence to be copied, a pair of short DNA molecules called primers, free DNA building blocks called deoxynucleotide triphosphates (dNTPs), and a specialized DNA polymerase enzyme. PCR involves a series of steps at high temperatures, requiring a DNA polymerase enzyme that is functional at such temperatures. The most commonly used DNA polymerase is Taq polymerase, named after Thermus aquaticus, the bacterium from which the polymerase was initially isolated. DNA

 Core: Biotechnology

RNA Interference

JoVE 10804

RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the expression of a gene by binding to its messenger RNA (mRNA) transcript, preventing the protein from being translated.

This process occurs naturally in cells, often through the activity of microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to selectively deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used to suppress genes that are overactive in diseases such as cancer. First, double-stranded RNA with a sequence complementary to the targeted gene is synthesized. Different types of double-stranded RNA can be used, including short interfering RNA (siRNA) and small hairpin RNA (shRNA). shRNA is one strand of RNA that is folded over—creating a double-stranded RNA with a hairpin loop on one side—and is a precursor of siRNA. The double-stranded RNA is then introduced into cells by methods such as injection or delivery by vectors, such as modified viruses. If shRNA is used, RNase enzymes in the cell, such as Dicer, cleave it down to the shorter siRNA, removing the hairpin loop. The siRNA then binds to an enzyme called Argonaute, which is part of a complex called RISC (RNA-induced silencing complex). Here, the two strands of the siRNA separate. One floats away w

 Core: Gene Expression

What is Gene Expression?

JoVE 10797

Gene expression is the process in which DNA (i.e., a gene) directs the synthesis of functional products, such as proteins. Cells can regulate gene expression at various stages. It allows organisms to generate different cell types and enables cells to adapt to internal and external factors.

A gene is a stretch of DNA that serves as the blueprint for functional RNAs and proteins. Since DNA is made up of nucleotides and proteins consist of amino acids, a mediator is required to convert the information that is encoded in DNA into proteins. This mediator is the messenger RNA (mRNA). mRNA copies the blueprint from DNA by a process called transcription. In eukaryotes, transcription takes place in the nucleus by complementary base-pairing with the DNA template. The mRNA is then processed and transported into the cytoplasm where it serves as a template for protein synthesis during translation. In prokaryotes, which lack a nucleus, the processes of transcription and translation occur at the same location and almost simultaneously since the newly-formed mRNA is susceptible to rapid degradation. Every cell of an organism contains the same DNA, and consequently the same set of genes. However, not all genes in a cell are “turned on” or use to synthesize proteins. A gene is said to be “expressed” when the protein it encodes is produced by the cel

 Core: Gene Expression

DNA Ligation Reactions

JoVE 5069

In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. An enzyme known as a ligase catalyzes the ligation reaction. In the cell, ligases repair single and double strand breaks that occur during DNA replication. In the laboratory, DNA ligase is used during molecular cloning to join DNA fragments of inserts with vectors…

 Basic Methods in Cellular and Molecular Biology

Types of RNA

JoVE 10800

Three main types of RNA are involved in protein synthesis: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). These RNAs perform diverse functions and can be broadly classified as protein-coding or non-coding RNA. Non-coding RNAs play important roles in the regulation of gene expression in response to developmental and environmental changes. Non-coding RNAs in prokaryotes can be manipulated to develop more effective antibacterial drugs for human or animal use. The central dogma of molecular biology states that DNA contains the information that encodes proteins and RNA uses this information to direct protein synthesis. Different types of RNA are involved in protein synthesis. Based on whether or not they encode proteins, RNA is broadly classified as protein-coding or non-coding RNA. Messenger RNA (mRNA) is the protein-coding RNA. It consists of codons—sequences of three nucleotides that encode a specific amino acid. Transfer RNA (tRNA) and ribosomal RNA (rRNA) are non-coding RNA. tRNA acts as an adaptor molecule that reads the mRNA sequence and places amino acids in the correct order in the growing polypeptide chain. rRNA and other proteins make up the ribosome—the seat of protein synthesis in the cell. During translation, ribosomes move along an mRNA strand where they stabilize the binding of tRNA molecules and catalyze the for

 Core: Gene Expression

Gene Silencing with Morpholinos

JoVE 5326

Morpholino-mediated gene silencing is a common technique used to study roles of specific genes during development. Morpholinos inhibit gene expression by hybridizing to complementary mRNAs. Due to their unique chemistry, morpholinos are easy to produce and store, which makes them remarkably cost effective compared to other gene silencing methods.


This video reviews proper…

 Developmental Biology

Zebrafish Microinjection Techniques

JoVE 5130

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage embryos. Using this technique, solutions containing genetic material or knockdown constructs are delivered into the blastomeres: the embryonic cells sitting atop the yolk of the newly fertilized egg. Delivery into the cytoplasm is…

 Biology II

RNAi in C. elegans

JoVE 5105

RNA interference (RNAi) is a widely used technique in which double stranded RNA is exogenously introduced into an organism, causing knockdown of a target gene. In the nematode, C. elegans, RNAi is particularly easy and effective because it can be delivered simply by feeding the worms bacteria that express double stranded RNA (dsRNA) that is complementary to a gene of interest. First,…

 Biology I

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

1Department of Molecular Cell Biology, University of California, Berkeley, 2Howard Hughes Medical Institute, University of California, Berkeley, 3Innovative Genomics Institute, University of California, Berkeley, 4Biomedical Sciences Graduate Program, University of California, San Francisco, 5Department of Microbiology and Immunology, University of California, San Francisco, 6Diabetes Center, University of California, San Francisco, 7Chan Zuckerberg Biohub, 8Department of Medicine, University of California, San Francisco, 9UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, 10Department of Integrative Biology, University of California, Berkeley

JoVE 57350

 Genetics

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

1Department of Biomedical Genetics, University of Rochester Medical Center, 2Department of Biostatistics and Computational Biology, University of Rochester Medical Center, 3Non-Clinical Statistics, Bristol-Myers Squibb, 4Department of Microbiology and Immunology, University of Rochester Medical Center

JoVE 57819

 Genetics

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

1Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, 2Department of Laboratory Medicine, National Taiwan University Hospital, 3Center for Microbial Pathogenesis, Nationwide Children's Hospital and the Department of Pediatrics, The Ohio State University

JoVE 57862

 Biochemistry

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

1Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Section on Nervous System Development and Plasticity, The Eunice Kennedy Shriver National Institute of Child and Human Development, National Institutes of Health, 3Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University, 4Department of Physiology, Anatomy and Genetics, University of Oxford

JoVE 55724

 Genetics

Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome

1Division of Hematology, Davidoff Cancer Center, Rabin Medical Center, 2The Center of Nanoscience and Nanotechnology, Tel Aviv University, 3Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, 4Department of Leukemia, The University of Texas MD Anderson Cancer Center

JoVE 53300

 Bioengineering
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