DNA Methylation

DNA methylation is a type of chemical modification of DNA that can be inherited and subsequently removed without changing the original DNA sequence. As such, it is part of the epigenetic code and is also the most well characterized epigenetic mechanism.
DNA methylation involves the addition of a methyl group to DNA — for example, to the number 5 carbon of the cytosine pyrimidine ring — with the effect of reducing gene expression. DNA methylation at the 5 position of cytosine has been found in every vertebrate examined. In adult somatic tissues, DNA methylation typically occurs in a CpG dinucleotide context; non-CpG methylation is prevalent in embryonic stem cells.
In plants, cytosines are methylated both symmetrically (CpG or CpNpG) and asymmetrically (CpNpNp), where N can be any nucleotide. Some organisms, such as fruit flies, exhibit virtually no DNA methylation

Metabolomics

Metabolomics is the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind" - specifically, the study of their small-molecule metabolite profiles.The metabolome represents the collection of all metabolites in a biological organism, which are the end products of its gene expression. Thus, while mRNA gene expression data and proteomic analyses do not tell the whole story of what might be happening in a cell, metabolic profiling can give an instantaneous snapshot of the physiology of that cell. One of the challenges of systems biology is to integrate proteomic, transcriptomic, and metabolomic information to give a more complete picture of living organisms.

Protein analysis

Protein analysis , and biomolecular NMR.contains a broad selection of products for Western blotting, protein electrophoresis, protein quantitation, protein chromatography, custom peptides, mass spectrometry, x-ray crystallography

Stem Cell Biology

Stem and progenitor cell research is a complex and very exciting field that promises fantastic curative discoveries in numerous areas from cancer to diabetes to neurogenerative diseases. Sigma-Aldrich offers an comprehensive and unprecedented number of products to support scientists in the discovery efforts in this area. These products are necessities in many areas including isolation, differentiation, genomics and epigenetics, functional profiling, and in vivo/in vitro tracking. Applications such as transfection, cell and protein characterization, RNAi, ADMET, and imaging are instrumental in the development of stem cells in regenerative medicine and drug discovery. The products contained within this Web site are considered tools for stem cell science and support our customers in their efforts to develop cardiac, hematopoietic, endocrine, and neurological disease therapies either in the areas of basic research, regenerative medicine, or drug efficacy and safety screening.

Proteomics

Proteomics research defines the dynamic nature of gene expression and regulation with detailed protein profiling, protein-protein interactions, and structural biology studies. Sigma's innovative technologies and products provide an integrated approach for proteomic analysis of both native and recombinant fusion proteins

Epigenetics

Epigenetics is the study of heritable changes in gene expression without a change in DNA sequence. The best understood Epigenetic mechanism, DNA methylation, refers to the addition of a methyl group by the enzyme DNA Methyltransferase to the 5-carbon of cytosine in a CpG dinucleotide. Methylation in regulatory regions adjacent to genes generally acts to suppress gene expression and/or regulation potentially having an impact on cellular function. Once a cell has an established DNA Methylation pattern, methlyated sites are inherited by daughter cells which have important implications in cellular function. Epigenetic research has demonstrated that aberrant DNA methylation is present in several disease states including cancer, in addition to other genetic diseases. Carcinogenesis can occur when DNA methylation acts to silence tumor suppressor genes, leading to heritable alterations of these genes.

DNA Replication

DNA replication is the process of copying a double-stranded DNA molecule to form two double-stranded molecules.The process of DNA replication is a fundamental process used by all living organisms as it is the basis for biological inheritance. As each DNA strand holds the same genetic information, both strands can serve as templates for the reproduction of the opposite strand. The template strand is preserved in its entirety and the new strand is assembled from nucleotides. This process is called "semiconservative replication". The resulting double-stranded DNA molecules are identical; proofreading and error-checking mechanisms exist to ensure near perfect fidelity.
In a cell, DNA replication must happen before cell division can occur. DNA synthesis begins at specific locations in the genome, called "origins", where the two strands of DNA are separated..RNA primers attach to single stranded DNA and the enzyme DNA polymerase extends the primers to form new strands of DNA, adding nucleotides matched to the template strand. The unwinding of DNA and synthesis of new strands forms a replication fork. In addition to DNA polymerase, a number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis.
DNA replication can also be performed artificially, using the same enzymes used within the cell. DNA polymerases and artificial DNA primers are used to initiate DNA synthesis at known sequences in a template molecule. The polymerase chain reaction (PCR), a common laboratory technique, employs artificial synthesis in a cyclic manner to rapidly and specifically amplify a target DNA fragment from a pool of DNA.

Cloning

A major problem in biochemical research is obtaining sufficient quantities of the substance of interest. These difficulties have been largely eliminated in recent years through the development of molecular cloning techniques. The clone is a collection of identical organisms that are all replicas of a single ancestor. Methods of creating clones of desired properties, usually called genetic engineering and recombinant DNA technology, deserve much of the credit for the dramatic rise of biotechnology since the mid-70'. The main idea of molecular cloning is to insert a DNA segment of interest into an autonomously replicating DNA molecule, called acloning vector, so that the DNA segment is replicated with the vector. Such vectors could be, for instance, plasmids (circular DNAs occuring in some bacteria). Reproduction of DNA segments in appropriate hosts, results in the production of large amount of the inserted DNA segment. A DNA to be cloned is usually a fragment of a genome of interest, obtained by application of restriction enzymes. Most restriction enzymes cleave duplex DNA at specific palindromic sites, generally two fragments that have single strand ends that are complimentary with each other (known as 'sticky ends'). Therefore, a restriction fragment can be inserted into a cut made in a cloning vector by the same restriction enzyme, because the segment ends stick (chemically bond) to the loose ends of the vector. Such a recombinant DNA molecule is inserted into a fast reproducing host cell, and is duplicated in the process of the host's reproduction. The cells containing the recombinant DNA are then isolated from non-infected cells using an antibiotic substance which the original vector is resistant to .

Sequencing

Sequencing is the operation of determining the nucleotide sequence of a given molecule. There are There are several approaches to sequencing, but generally, the most successful is based on gel electrophoresis. As mentioned earlier, the DNA polymerase enzyme catalyzes the replication reaction of DNA. DNA polymerase extends the chain by adding nucleotides to its end. Current biotechnology enables synthesis of nucleotides which cause the strand to terminate. For instance, A* denotes an Adenine molecule which does not allow other molecules to extend the strand after itself. By catalyzing DNA replication in an environment containing mixtures of normal Adenine and sythesized Adenine* instead of only Adenine, it is possible to create DNA strands of different lengths. By applying gel electrophoresis to these molecules, it is possible to determine the lengths of all the strings and from it to conclude the location of all Adenines in the tested DNA strand. In a similar fashion it is possible to locate other nucleotides and eventually to fully sequence a whole segment of DNA. Using this method, sequences of 500-800 nucleotides can be mapped.

Gel Electrophoresis

Gel electrophoresis is a technique used to separate a mixture of digested DNA fragments. An electrical field is used to move the negatively charged DNA molecules through porous agarose gel. Fragments of the same size and shape move at the same speed, and because smaller molecules travel faster then larger molecules, the mixture is separated into bands.
The amount of exposure the DNA receives to restriction enzymes determines the portion of possible sites that were actually separated. Therefore, by applying different exposures to the same DNA sequence, we can measure all possible lengths of DNA fragments, that one can obtain using a particular enzyme. From this information we can attempt to find out where the sites are located in the original molecule. This problem is known as:

Restriction Enzymes

One of the basic tools used in biotechnology is restriction enzymes. In natural circumstances, one of the main roles of these enzymes is to break foreign DNA entering the cell. A restriction enzyme breaks the phosphodiester bonds of a DNA upon appearance of a certain cleavage sequence. Each such enzyme is characterized by a different cleavage sequence. Today there are more then 150 known different cleavage sites, namely, different nucleotide configurations that known enzymes can digest.