DNA Microarray

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 (see uses and types section). Microarrays also differ in fabrication, workings, accuracy, efficiency, and cost (see fabrication section). Additional factors for microarray experiments are the experimental design and the methods of analyzing the data.

DNA Modifying Enzymes

Nucleases are enzymes that cut DNA strands by catalyzing the hydrolysis of the phosphodiester bonds. Nucleases that hydrolyse nucleotides from the ends of DNA strands are called exonucleases, while endonucleases cut within strands. The most frequently used nucleases in molecular biology are the restriction endonucleases, which cut DNA at specific sequences. For instance, the EcoRV enzyme shown to the left recognizes the 6-base sequence 5′-GAT|ATC-3′ and makes a cut at the vertical line. In nature, these enzymes protect bacteria against phage infection by digesting the phage DNA when it enters the bacterial cell, acting as part of the restriction modification system. In technology, these sequence-specific nucleases are used in molecular cloning and DNA fingerprinting. Enzymes called DNA ligases can rejoin cut or broken DNA strands. Ligases are particularly important in lagging strand DNA replication, as they join together the short segments of DNA produced at the replication fork into a complete copy of the DNA template. They are also used in DNA repair and genetic recombination.

DNA Binding proteins

Structural proteins that bind DNA are well-understood examples of non-specific DNA-protein interactions. Within chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact structure called chromatin. In eukaryotes this structure involves DNA binding to a complex of small basic proteins called histones, while in prokaryotes multiple types of proteins are involved. The histones form a disk-shaped complex called a nucleosome, which contains two complete turns of double-stranded DNA wrapped around its surface. These non-specific interactions are formed through basic residues in the histones making ionic bonds to the acidic sugar-phosphate backbone of the DNA, and are therefore largely independent of the base sequence. Chemical modifications of these basic amino acid residues include methylation, phosphorylation and acetylation. These chemical changes alter the strength of the interaction between the DNA and the histones, making the DNA more or less accessible to transcription factors and changing the rate of transcription. Other non-specific DNA-binding proteins in chromatin include the high-mobility group proteins, which bind to bent or distorted DNA.These proteins are important in bending arrays of nucleosomes and arranging them into the larger structures that make up chromosomes.

A distinct group of DNA-binding proteins are the DNA-binding proteins that specifically bind single-stranded DNA. In humans, replication protein A is the best-understood member of this family and is used in processes where the double helix is separated, including DNA replication, recombination and DNA repair. These binding proteins seem to stabilize single-stranded DNA and protect it from forming stem-loops or being degraded by nucleases.

DNA Replication

Cell division is essential for an organism to grow, but when a cell divides it must replicate the DNA in its genome so that the two daughter cells have the same genetic information as their parent. The double-stranded structure of DNA provides a simple mechanism for DNA replication. Here, the two strands are separated and then each strand's complementary DNA sequence is recreated by an enzyme called DNA polymerase. This enzyme makes the complementary strand by finding the correct base through complementary base pairing, and bonding it onto the original strand. As DNA polymerases can only extend a DNA strand in a 5′ to 3′ direction, different mechanisms are used to copy the antiparallel strands of the double helix. In this way, the base on the old strand dictates which base appears on the new strand, and the cell ends up with a perfect copy of its DNA.

Alternate DNA structures

DNA exists in many possible conformations that include A-DNA, B-DNA, and Z-DNA forms, although, only B-DNA and Z-DNA have been directly observed in functional organisms. The conformation that DNA adopts depends on the hydration level, DNA sequence, the amount and direction of supercoiling, chemical modifications of the bases, the type and concentration of metal ions, as well as the presence of polyamines in solution.

The first published reports of A-DNA X-ray diffraction patterns— and also B-DNA used analyses based on Patterson transforms that provided only a limited amount of structural information for oriented fibers of DNA. An alternate analysis was then proposed by Wilkins et al., in 1953, for the in vivo B-DNA X-ray diffraction/scattering patterns of highly hydrated DNA fibers in terms of squares of Bessel functions. In the same journal, Watson and Crick presented their molecular modeling analysis of the DNA X-ray diffraction patterns to suggest that the structure was a double-helix.

Although the `B-DNA form' is most common under the conditions found in cells, it is not a well-defined conformation but a family of related DNA conformations that occur at the high hydration levels present in living cells. Their corresponding X-ray diffraction and scattering patterns are characteristic of molecular paracrystals with a significant degree of disorder.

DNA Properties

DNA is a long polymer made from repeating units called nucleotides. The DNA chain is 22 to 26 Ångströms wide (2.2 to 2.6 nanometres), and one nucleotide unit is 3.3 Å (0.33 nm) long.Although each individual repeating unit is very small, DNA polymers can be very large molecules containing millions of nucleotides. For instance, the largest human chromosome, chromosome number 1, is approximately 220 million base pairs long.

In living organisms, DNA does not usually exist as a single molecule, but instead as a pair of molecules that are held tightly together. These two long strands entwine like vines, in the shape of a double helix. The nucleotide repeats contain both the segment of the backbone of the molecule, which holds the chain together, and a base, which interacts with the other DNA strand in the helix. A base linked to a sugar is called a nucleoside and a base linked to a sugar and one or more phosphate groups is called a nucleotide. If multiple nucleotides are linked together, as in DNA, this polymer is called a polynucleotide.

The backbone of the DNA strand is made from alternating phosphate and sugar residues.The sugar in DNA is 2-deoxyribose, which is a pentose (five-carbon) sugar. The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These asymmetric bonds mean a strand of DNA has a direction. In a double helix the direction of the nucleotides in one strand is opposite to their direction in the other strand: the strands are antiparallel. The asymmetric ends of DNA strands are called the 5′ (five prime) and 3′ (three prime) ends, with the 5' end having a terminal phosphate group and the 3' end a terminal hydroxyl group. One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced by the alternative pentose sugar ribose in RNA.

DNA structure and function

It is not always the case that the structure of a molecule is easy to relate to its function. What makes the structure of DNA so obviously related to its function was described modestly at the end of the article: "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material".

The "specific pairing" is a key feature of the Watson and Crick model of DNA, the pairing of nucleotide subunits. In DNA, the amount of guanine is equal to cytosine and the amount of adenine is equal to thymine. The A:T and C:G pairs are structurally similar. In particular, the length of each base pair is the same and they fit equally between the two phosphate backbones . The base pairs are held together by hydrogen bonds, a type of chemical attraction that is easy to break and easy to reform. After realizing the structural similarity of the A:T and C:G pairs, Watson and Crick soon produced their double helix model of DNA with the hydrogen bonds at the core of the helix providing a way to unzip the two complementary strands for easy replication: the last key requirement for a likely model of the genetic molecule.

Indeed, the base-pairing did suggest a way to copy a DNA molecule. Just pull apart the two phosphate backbones, each with its hydrogen bonded A, T, G, and C components. Each strand could then be used as a template for assembly of a new base-pair complementary strand.

Paracrystalline lattice models of B-DNA structures

A paracrystalline lattice, or paracrystal, is a molecular or atomic lattice with significant amounts (e.g., larger than a few percent) of partial disordering of molecular arranegements. Limiting cases of the paracrystal model are nanostructures, such as glasses, liquids, etc., that may possess only local ordering and no global order. A simple example of a paracrystalline lattice is shown in the following figure for a silica glass: Liquid crystals also have paracrystalline rather than crystalline structures.

Highly hydrated B-DNA occurs naturally in living cells in such a paracrystalline state, which is a dynamic one in spite of the relatively rigid DNA double-helix stabilized by parallel hydrogen bonds between the nucleotide base-pairs in the two complementary, helical DNA chains (see figures). For simplicity most DNA molecular models ommit both water and ions dynamically bound to B-DNA, and are thus less useful for understanding the dynamic behaviors of B-DNA in vivo. The physical and mathematical analysis of X-ray and spectroscopic data for paracrystalline B-DNA is therefore much more complicated than that of crystalline, A-DNA X-ray diffraction patterns. The paracrystal model is also important for DNA technological applications such as DNA nanotechnology. Novel techniques that combine X-ray diffraction of DNA with X-ray microscopy in hydrated living cells are now also being developed.

DNA structure

The structure of DNA shows a variety of forms, both double-stranded and single-stranded. The mechanical properties of DNA, which are directly related to its structure, are a significant problem for cells. Every process which binds or reads DNA is able to use or modify the mechanical properties of DNA for purposes of recognition, packaging and modification. The extreme length (a chromosome may contain a 10 cm long DNA strand), relative rigidity and helical structure of DNA has led to the evolution of histones and of enzymes such as topoisomerases and helicases to manage a cell's DNA. The properties of DNA are closely related to its molecular structure and sequence, particularly the weakness of the hydrogen bonds and electronic interactions that hold strands of DNA together compared to the strength of the bonds within each strand.

Experimental techniques which can directly measure the mechanical properties of DNA are relatively new, and high-resolution visualization in solution is often difficult. Nevertheless, scientists have uncovered large amount of data on the mechanical properties of this polymer, and the implications of DNA's mechanical properties on cellular processes is a topic of active current research.

The DNA found in many cells can be macroscopic in length - a few centimetres long for each human chromosome. Consequently, cells must compact or "package" DNA to carry it within them. In eukaryotes this is carried by spool-like proteins known as histones, around which DNA winds. It is the further compaction of this DNA-protein complex which produces the well known mitotic eukaryotic chromosomes.

Molecular models of DNA

Molecular models of DNA structures are representations of the molecular geometry and topology of Deoxyribonucleic acid (DNA) molecules using one of several means, with the aim of simplifying and presenting the essential, physical and chemical, properties of DNA molecular structures either in vivo or in vitro. These representations include closely packed spheres (CPK models) made of plastic, metal wires for 'skeletal models', graphic computations and animations by computers, artistic rendering. Computer molecular models also allow animations and molecular dynamics simulations that are very important for understanding how DNA functions in vivo.

The more advanced, computer-based molecular models of DNA involve molecular dynamics simulations as well as quantum mechanical computations of vibro-rotations, delocalized molecular orbitals (MOs), electric dipole moments, hydrogen-bonding, and so on. DNA molecular dynamics modeling involves simulations of DNA molecular geometry and topology changes with time as a result of both intra- and inter- molecular interactions of DNA. Whereas molecular models of Deoxyribonucleic acid (DNA) molecules such as closely packed spheres (CPK models) made of plastic or metal wires for 'skeletal models' are useful representations of static DNA structures, their usefulness is very limited for representing complex DNA dynamics. Computer molecular modeling allows both animations and molecular dynamics simulations that are very important for understanding how DNA functions in vivo.