Gel electrophoresis is a method for separation and analysis of biomacromolecules and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.
Molecular biology is a branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions.
The northern blot, or RNA blot, is a technique used in molecular biology research to study gene expression by detection of RNA in a sample.
In genetics and biochemistry, sequencing means to determine the primary structure of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succinctly summarizes much of the atomic-level structure of the sequenced molecule.
Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Electrophoretic mobility is a function of the length, conformation, and charge of the molecule. Polyacrylamide gel electrophoresis is a powerful tool used to analyze RNA samples. When polyacrylamide gel is denatured after electrophoresis, it provides information on the sample composition of the RNA species.
Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small fragments of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, molecular cloning and as molecular probes. In nature, oligonucleotides are usually found as small RNA molecules that function in the regulation of gene expression, or are degradation intermediates derived from the breakdown of larger nucleic acid molecules.
Polyacrylamide (abbreviated as PAM or pAAM) is a polymer with the formula (-CH2CHCONH2-). It has a linear-chain structure. PAM is highly water-absorbent, forming a soft gel when hydrated. In 2008, an estimated 750,000,000 kg were produced, mainly for water treatment and the paper and mineral industries.
A protein microarray is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale. Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel. The chip consists of a support surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, to which an array of capture proteins is bound. Probe molecules, typically labeled with a fluorescent dye, are added to the array. Any reaction between the probe and the immobilised protein emits a fluorescent signal that is read by a laser scanner. Protein microarrays are rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents. The concept and methodology of protein microarrays was first introduced and illustrated in antibody microarrays in 1983 in a scientific publication and a series of patents. The high-throughput technology behind the protein microarray was relatively easy to develop since it is based on the technology developed for DNA microarrays, which have become the most widely used microarrays.
Aptamers are short sequences of artificial DNA, RNA, XNA, or peptide that bind a specific target molecule, or family of target molecules. They exhibit a range of affinities, with variable levels of off-target binding and are sometimes classified as chemical antibodies. Aptamers and antibodies can be used in many of the same applications, but the nucleic acid-based structure of aptamers, which are mostly oligonucleotides, is very different from the amino acid-based structure of antibodies, which are proteins. This difference can make aptamers a better choice than antibodies for some purposes.
Systematic evolution of ligands by exponential enrichment (SELEX), also referred to as in vitro selection or in vitro evolution, is a combinatorial chemistry technique in molecular biology for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to a target ligand or ligands. These single-stranded DNA or RNA are commonly referred to as aptamers. Although SELEX has emerged as the most commonly used name for the procedure, some researchers have referred to it as SAAB and CASTing SELEX was first introduced in 1990. In 2015, a special issue was published in the Journal of Molecular Evolution in the honor of quarter century of the discovery of SELEX.
SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common types of genetic variation. An SNP is a single base pair mutation at a specific locus, usually consisting of two alleles. SNPs are found to be involved in the etiology of many human diseases and are becoming of particular interest in pharmacogenetics. Because SNPs are conserved during evolution, they have been proposed as markers for use in quantitative trait loci (QTL) analysis and in association studies in place of microsatellites. The use of SNPs is being extended in the HapMap project, which aims to provide the minimal set of SNPs needed to genotype the human genome. SNPs can also provide a genetic fingerprint for use in identity testing. The increase of interest in SNPs has been reflected by the furious development of a diverse range of SNP genotyping methods.
Cell-free protein array technology produces protein microarrays by performing in vitro synthesis of the target proteins from their DNA templates. This method of synthesizing protein microarrays overcomes the many obstacles and challenges faced by traditional methods of protein array production that have prevented widespread adoption of protein microarrays in proteomics. Protein arrays made from this technology can be used for testing protein–protein interactions, as well as protein interactions with other cellular molecules such as DNA and lipids. Other applications include enzymatic inhibition assays and screenings of antibody specificity.
Nucleic acid methods are the techniques used to study nucleic acids: DNA and RNA.
Integrated DNA Technologies, Inc. (IDT), headquartered in Coralville, Iowa, is a supplier of custom nucleic acids, serving the areas of academic research, biotechnology, clinical diagnostics, and pharmaceutical development. IDT's primary business is the manufacturing of custom DNA and RNA oligonucleotides (oligos) for research applications.
MAGIChips, also known as "microarrays of gel-immobilized compounds on a chip" or "three-dimensional DNA microarrays", are devices for molecular hybridization produced by immobilizing oligonucleotides, DNA, enzymes, antibodies, and other compounds on a photopolymerized micromatrix of polyacrylamide gel pads of 100x100x20µm or smaller size. This technology is used for analysis of nucleic acid hybridization, specific binding of DNA, and low-molecular weight compounds with proteins, and protein-protein interactions.
DNA-encoded chemical libraries (DECL) is a technology for the synthesis and screening on an unprecedented scale of collections of small molecule compounds. DECL is used in medicinal chemistry to bridge the fields of combinatorial chemistry and molecular biology. The aim of DECL technology is to accelerate the drug discovery process and in particular early phase discovery activities such as target validation and hit identification.
Molecular Inversion Probe (MIP) belongs to the class of Capture by Circularization molecular techniques for performing genomic partitioning, a process through which one captures and enriches specific regions of the genome. Probes used in this technique are single stranded DNA molecules and, similar to other genomic partitioning techniques, contain sequences that are complementary to the target in the genome; these probes hybridize to and capture the genomic target. MIP stands unique from other genomic partitioning strategies in that MIP probes share the common design of two genomic target complementary segments separated by a linker region. With this design, when the probe hybridizes to the target, it undergoes an inversion in configuration and circularizes. Specifically, the two target complementary regions at the 5’ and 3’ ends of the probe become adjacent to one another while the internal linker region forms a free hanging loop. The technology has been used extensively in the HapMap project for large-scale SNP genotyping as well as for studying gene copy alterations and characteristics of specific genomic loci to identify biomarkers for different diseases such as cancer. Key strengths of the MIP technology include its high specificity to the target and its scalability for high-throughput, multiplexed analyses where tens of thousands of genomic loci are assayed simultaneously.
Polony sequencing is an inexpensive but highly accurate multiplex sequencing technique that can be used to “read” millions of immobilized DNA sequences in parallel. This technique was first developed by Dr. George Church's group at Harvard Medical School. Unlike other sequencing techniques, Polony sequencing technology is an open platform with freely downloadable, open source software and protocols. Also, the hardware of this technique can be easily set up with a commonly available epifluorescence microscopy and a computer-controlled flowcell/fluidics system. Polony sequencing is generally performed on paired-end tags library that each molecule of DNA template is of 135 bp in length with two 17–18 bp paired genomic tags separated and flanked by common sequences. The current read length of this technique is 26 bases per amplicon and 13 bases per tag, leaving a gap of 4–5 bases in each tag.
Illumina dye sequencing is a technique used to determine the series of base pairs in DNA, also known as DNA sequencing. The reversible terminated chemistry concept was invented by Bruno Canard and Simon Sarfati at the Pasteur Institute in Paris. It was developed by Shankar Balasubramanian and David Klenerman of Cambridge University, who subsequently founded Solexa, a company later acquired by Illumina. This sequencing method is based on reversible dye-terminators that enable the identification of single nucleotides as they are washed over DNA strands. It can also be used for whole-genome and region sequencing, transcriptome analysis, metagenomics, small RNA discovery, methylation profiling, and genome-wide protein-nucleic acid interaction analysis.
A bridged nucleic acid (BNA) is a modified RNA nucleotide. They are sometimes also referred to as constrained or inaccessible RNA molecules. BNA monomers can contain a five-membered, six-membered or even a seven-membered bridged structure with a "fixed" C3'-endo sugar puckering. The bridge is synthetically incorporated at the 2', 4'-position of the ribose to afford a 2', 4'-BNA monomer. The monomers can be incorporated into oligonucleotide polymeric structures using standard phosphoramidite chemistry. BNAs are structurally rigid oligo-nucleotides with increased binding affinities and stability.
