DNA microarray

A DNA microarray (also commonly known as gene chip, DNA chip, or biochip) is a collection of microscopic DNA spots attached to a solid surface.Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome.
A DNA microarray is a multiplex technology used in molecular biology and in Medicine. It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing picomoles (10⁻¹² moles) of a specific DNA sequence, known as probes (or reporters). This can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target. Since an array can contain tens of thousands of probes, a microarray experiment can accomplish many genetic tests in parallel. Therefore arrays have dramatically accelerated many types of investigation.

In standard microarrays, the probes are attached via surface engineering to a solid surface by a covalent bond to a chemical matrix (via epoxy-silane, amino-silane, lysine, polyacrylamide or others). The solid surface can be glass or a silicon chip, in which case they are colloquially known as an Affy chip when an Affymetrix chip is used. Other microarray platforms, such as Illumina, use microscopic beads, instead of the large solid support. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system.

DNA microarrays can be used to measure changes in expression levels, to detect single nucleotide polymorphisms (SNPs) , to genotype or resequence mutant genomes.

Denaturation

Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), or heat. If proteins in a living cell are denatured, this results in disruption of cell activity and possibly cell death. Denatured proteins can exhibit a wide range of characteristics, from loss of solubility to communal aggregation. Denaturization in this sense is not used in preparing the industrial chemical denatured alcohol.

Denaturation is the alteration of a protein shape through some form of external stress (for example, by applying heat, acid or alkali), in such a way that it will no longer be able to carry out its cellular function.

Denatured proteins can exhibit a wide range of characteristics, from loss of solubility to communal aggregation.

Proteins are very long strands of amino acids linked together in specific sequences.

A protein is created by ribosomes that "read" codons in the gene and assemble the requisite amino acid combination from the genetic instruction, in a process known as translation.

The newly created protein strand then undergoes post-translational modification in which additional atoms or molecules are added, for example copper, zinc, iron.

Once this post-translational modification process has been completed, the protein begins to fold (spontaneously, and sometimes with enzymatic assistance), curling up on itself so that hydrophobic elements of the protein are buried deep inside the structure and hydrophilic elements end up on the outside.

The final shape of a protein determines how it interacts with its environment.

Plasmid Isolation (Alkaline Lysis)

Bacterial plasmids, the non-genomic transferable DNA, can easily be purified from bacteria using numerous techniques. The purification of DNA is important for genetic research as it provides a source of transferable DNA and allows researchers to isolate large amounts of recombinant DNA. One common technique for plasmid purification is the alkaline lysis method, which breaks open bacteria with an alkaline solution, proteins are removed by precipitation and the plasmid DNA is recovered with alcohol precipitation.

Students purify bacterial plasmids from a liquid culture using this alkaline lysis method.

Protein Function

Proteins are very important molecules in our cells. They are involved in virtually all cell functions. Each protein within the body has a specific function. Some proteins are involved in structural support, while others are involved in bodily movement, or in defense against germs.

Proteins vary in structure as well as function. They are constructed from a set of 20 amino acids and have distinct three-dimensional shapes. Below is a list of several types of proteins and their functions.

Protein Functions

Antibodies - are specialized proteins involved in defending the body from antigens (foreign invaders). One way antibodies destroy antigens is by immobilizing them so that they can be destroyed by white blood cells.

Contractile Proteins - are responsible for movement. Examples include actin and myosin. These proteins are involved in muscle contraction and movement.

Enzymes - are proteins that facilitate biochemical reactions. They are often referred to as catalysts because they speed up chemical reactions. Examples include the enzymes lactase and pepsin. Lactase breaks down the sugar lactose found in milk. Pepsin is a digestive enzyme that works in the stomach to break down proteins in food.

Hormonal Proteins - are messenger proteins which help to coordinate certain bodily activities. Examples include insulin, oxytocin, and somatotropin. Insulin regulates glucose metabolism by controlling the blood-sugar concentration. Oxytocin stimulates contractions in females during childbirth. Somatotropin is a growth hormone that stimulates protein production in muscle cells.

Structural Proteins - are fibrous and stringy and provide support. Examples include keratin, collagen, and elastin. Keratins strengthen protective coverings such as hair, quills, feathers, horns, and beaks. Collagens and elastin provide support for connective tissues such as tendons and ligaments.

Storage Proteins - store amino acids. Examples include ovalbumin and casein. Ovalbumin is found in egg whites and casein is a milk-based protein.

Transport Proteins - are carrier proteins which move molecules from one place to another around the body. Examples include hemoglobin and cytochromes. Hemoglobin transports oxygen through the blood. Cytochromes operate in the electron transport chain as electron carrier proteins.

Polymers

A polymer is a substance with a high molecular mass that is composed of a large number of repeating units. These units, called monomers, are connected by covalent chemical bonds.

Some polymers are composed of a single type of monomer, while others may consist of two, three or more different monomers. Many biological macromolecules are examples of natural polymers. These include the carbohydrates, starch, cellulose and glycogen (branched chains of glucose monomers), and chitin (chains of N-acetyl-glucosamine). Examples of polymers consisting of mixtures of monomers are the nucleic acids, DNA and RNA, made from units of 4 different nucleotides, and proteins, which consist of a mixture of the 20 standard amino acids. Natural rubber, or latex, is a natural hydrocarbon polymer found in the sap of some plants. Natural, biological polymers have both structural roles and physiological functions, and are involved in the control of cellular operations such as growth, replication and metabolism.

Synthetic polymers can be produced commercially, and are traditionally derived from petroleum products. They have a wide variety of properties and uses. The most common synthetic polymers are plastics such as polyethylene and nylon. Synthetic polymers made out of glycolic and lactic acids, and other biodegradable materials, have become increasingly popular for use in biomedical applications. Man-made polymers that react to their surroundings are known as smart polymers, or stimulus-responsive polymers, and can be used for a variety of purposes in technology and biomedicine.

DNA Transcription

DNA transcription is a process that involves the transcribing of genetic information from DNA to RNA.

DNA is housed within the nucleus of our cells. It controls cellular activity by coding for the production of enzymes and proteins. The information in DNA is not directly converted into proteins, but must first be copied into RNA. This ensures that the information contained in the DNA does not become tainted.

DNA Transcription

DNA consists of four nucleotide bases [adenine (A), guanine (G), cytosine (C) and thymine (T)] that are paired together (A-T and C-G) to give DNA its double helical shape.

DNA is transcribed by an enzyme called RNA polymerase. Specific nucleotide sequences tell RNA polymerase where to begin and where to end. RNA polymerase attaches to the DNA at a specific area called the promoter region. The DNA strand opens and allows RNA polymerase to transcribe only a single strand of DNA into a single stranded RNA polymer called messenger RNA (mRNA).

Like DNA, RNA is composed of nucleotide bases. RNA however, contains the nucleotides adenine, guanine, cytosine and uricil (U). When RNA polymerase transcribes the DNA, guanine pairs with cytosine and adenine pairs with uricil. RNA polymerase moves along the DNA until it reaches a terminator sequence. At that point, RNA polymerase releases the mRNA polymer and detaches from the DNA.

Since proteins are constructed in the cytoplasm of the cell by a process called translation, mRNA must cross the nuclear membrane to reach the cytoplasm. Once in the cytoplasm, mRNA along with ribosomes and another RNA molecule called transfer RNA, work together to produce proteins. Proteins can be manufactured in large quantities because a single DNA sequence can be transcribed by many RNA polymerase molecules at once.