DNA polymerase

A DNA polymerase is an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best-known for their role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand. This process copies a piece of DNA. The newly-polymerized molecule is complementary to the template strand and identical to the template's original partner strand. DNA polymerases use a magnesium ion for catalytic activity.

Polymerase chain reaction

The polymerase chain reaction (PCR) is a technique in molecular biology to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.

DNA microarray

A DNA microarray is a multiplex technology used in molecular biology. 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.

Oligonucleotide

An oligonucleotide (from Greek prefix oligo-, "having few, having little") is a short nucleic acid polymer, typically with twenty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized by polymerizing individual nucleotide precursors. Automated synthesizers allow the synthesis of oligonucleotides up to 160 to 200 bases.

The length of the oligonucleotide is usually denoted by "mer" (from Greek meros, "part"). For example, a fragment of 25 bases would be called a 25-mer. Because oligonucleotides readily bind to their respective complementary nucleotide, they are often used as probes for detecting DNA or RNA. Examples of procedures that use oligonucleotides include DNA microarrays, Southern blots, ASO analysis, fluorescent in situ hybridization (FISH), and the synthesis of artificial genes.

Oligonucleotides composed of DNA (oligodeoxyribonucleotides) are often used in the polymerase chain reaction, a procedure that can greatly amplify almost any small piece of DNA. There, the oligonucleotide is referred to as a primer, allowing DNA polymerase to extend the oligonucleotide and replicate the complementary strand.

Polymer

A polymer is a large molecule (macromolecule) composed of repeating structural units typically connected by covalent chemical bonds. While polymer in popular usage suggests plastic, the term actually refers to a large class of natural and synthetic materials with a wide variety of properties.

Because of the extraordinary range of properties accessible in polymeric materials, they play an essential and ubiquitous role in everyday life, ranging from familiar synthetic plastics and elastomers to natural biopolymers such as DNA and proteins that are essential for life. A simple example is polyethylene, whose repeating unit is based on ethylene (IUPAC name ethene) monomer. Most commonly, as in this example, the continuously linked backbone of a polymer used for the preparation of plastics consists mainly of carbon atoms. However, other structures do exist; for example, elements such as silicon form familiar materials such as silicones, examples being silly putty and waterproof plumbing sealant. The backbone of DNA is in fact based on a phosphodiester bond, and repeating units of polysaccharides (e.g. cellulose) are joined together by glycosidic bonds via oxygen atoms.

Complementary DNA

In genetics, complementary DNA (cDNA) is DNA synthesized from a mature mRNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes. When scientists want to express a specific protein in a cell that does not normally express that protein (i.e., heterologous expression), they will transfer the cDNA that codes for the protein to the recipient cell. cDNA is also produced by retroviruses (such as HIV-1, HIV-2, Simian Immunodeficiency Virus, etc.) which is integrated into its host to create a provirus.

Gel electrophoresis

Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric field applied to a gel matrix. DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR, but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization.

Illumina Methylation Assay

The Illumina Methylation Assay using the Infinium II platform uses "BeadChip" technology to generate a comprehensive genome wide profiling of human DNA methylation. Similar to bisulfite sequencing and pyrosequencing, this method quantifies methylation levels at specific loci within the genome. Although the assay does not encompass the entire human genome, it can measure methylation level at 27,578 CpG dinucloeotides in 14,495 genes.

Allele Specific Oligonucleotide

Allele specific oligonucleotide (ASO) is a technique that allows detection of single base mutations without the need for PCR or gel electrophoresis. Short (20-25 nucleotides in length), labeled probes are exposed to the non-fragmented target DNA. Hybridization occurs with high specificity due to the short length of the probes and even a single base change will hinder hybridization. The target DNA is then washed and the labeled probes that didn't hybridize are removed. The target DNA is then analyzed for the presence of the probe via radioactivity or fluorescence. In this experiment, as in most molecular biology techniques, a control must be used to ensure successful experimentation. The Illumina Methylation Assay is an example of a method that takes advantage of the ASO technique to measure one base pair differences in sequence.