Microarray approaches to finding protein–DNA binding

Mapping protein localization to DNA using chromatin immunoprecipitation and DNA microarrays^32, 33. In this technique, proteins are crosslinked to genomic DNA in a cellular context. The DNA is sheared or digested, leaving DNA–protein complexes that can be precipitated using protein-specific antibodies. Two pools of DNA fragment — those released from the protein and those from control DNA — are amplified and labelled using different fluorescent dyes. Both sets of probes are simultaneously hybridized to a DNA microarray that contains intragenic DNA sequences. The greater the difference in the fluorescent intensity at any fragment on the array, the stronger the binding of the protein to that fragment. b | Mapping protein localization to DNA using DNA adenine methyltransferase identification³⁴. In this method, the DNA adenine methyltransferase enzyme is fused to a chromatin-associated protein and is expressed in cells; the chimeric protein binds to chromatin and methylates adenine residues in the vicinity of the protein-binding site. The methylation-specific restriction enzyme Dpn1 recognizes and cleaves the DNA at methylated GATC sites. The resulting fragments are fractionated by size, then labelled and probed to the array.

Technologies MicroArray

2 samples of RNA are taken from 2 seperate sources and cDNA is created from each by using a technique called RT-PCR (Reverse Transcriptase Polymerase Chain Reaction). Each cDNA sample is labeled with its own specific dye. Both cDNA samples are then washed over a single array, after which dye intensities on each spot is calculated using a fluorescence camera.
Aging
-The free radical theory of aging
-Mitochondria and aging
-The glycation theory of aging
-Proteins damage and maintenance in aging
-Dna damage and dna repair
-Telomeres and aging
-Cellular senescence and apoptosis in aging
-Longevity genes
-Gene silencing in aging
-Hormones and aging
-The immune system and aging
-Inflammation and aging
-Accumulation of toxins and chemical garbage
-Cancer and aging
-Biomarkers of aging
-Caloric restriction with adequate nutrition

Detailed cDNA Microarray Technology Scheme

DNA microarrays work on the principal of base-pairing (See: Basic Biology, base-pairing). Base-pairing allows probes to hybridize to targets on the microarray. (See Basic Biology, hybridization).

At a basic level microarrays are implemented as follows: a cell's RNA is extracted. This RNA (targets) is then multiplied, labeled with fluorescence and hybridized to existing DNA (probes) on the microarray. After hybridization, the probes that were hybridized with targets are fluorescent and a computer scanner is able to detect this fluorescence. Those probes that are fluorescent correspond to the genes that were expressed in the cell.

The microarray is composed of millions of spots (sometimes referred to as cells), each with thousands of probes. Each 20 micrometer cell can contain up to 10^7 probes. The enormous number of probes is to increase hybridization probability and possibilities. Figure 1 is an illustration of the principal of hybridization. A cell (G) is laced with probes of DNA (either oligonucleotide sequences, or cDNA. See below for more information). The fluorescent targets (green circles) are then exposed to the microarray and allowed to hybridize.

Experiments using cDNA microarrays typically involve two cells: a control cell and an experimental cell. First, using robotics, the microarray is laced with DNA probes corresponding to the genes of interest (or the entire organism's genome).

Second, mRNA from a control cell and an experimental cell is isolated. This mRNA corresponds to the genes in the cell that are being expressed (See basic biology, Why study genes). Using reverse transcriptase , the mRNA is then converted to cDNA . The cDNA from both cells are labeled, using fluorescence, different colors. This fluorescent dye can be identified by a computer scanner. The labeled cDNA is considered the target, which hybridizes via base-pair interactions with the probe. (See Basic Biology: Hybridization)

Once the targets are exposed to the microarray for a sufficient amount of time to allow for hybridization (typically 12-16 hours), the array is washed. Certain probes on the array will now be fluorescent, because the fluorescent targets have hybridized to them.

Only the probes containing genes that have been transcribed in the cell will be fluorescent. Those genes that are being expressed in the control cell will fluoresce one color (green), while those expressed in the experimental cell will fluoresce another color (red). Those genes expressed in both cells will have a mixed color (yellow). The amount each gene is being expressed can also be measured by how intense the fluorescence is.

A computer is used to measure the intensity and color of each and every spot on the microarray. Software can then be used to produce data on exactly which genes are being expressed in the cells, and how much each of those genes is being expressed.

Oligonucleotide Arrays

Oligonucleotide arrays are commonly used in biology laboratories and in clinical research projects. The Affymetrix GeneChip is the most widely used oligonucleotide array. Whereas the aforementioned cDNA microarrays use long strands of DNA as fixed probes, the GeneChip uses oligonucleotide sequences as its probe. The whole genome of an organism can be placed on a single microarray as oligonucleotide probes. These oligonucleotide sequences are usually around 25 base pairs in length (see Basic Biology: Base Pairs) (Ref. #7). Below is a explanation of how Affymetrix's technology works.

In order to use the array, first mRNA is extracted from a cell and reverse transcriptase is applied to obtain cDNA. In oligonucleotide arrays, in vitro transcription (for more information on in vitro transcription, click here) occurs to obtain biotin labeled cRNA (See Basic Biology: Base Pairing with RNA). These cRNA molecules are then exposed to the microarray. Overnight, the cRNA molecules (the targets) hybridize to the oligonucleotide probes. After hybridization, the chip is stained with a fluorescent molecule (streptavidin-phycoerythrin) that binds to biotin. The staining protocol includes a signal amplification step that employs anti-Streptavidin antibody and biotinylated goat IgG antibody (The series of washes and stains with aforementioned reagents binds the biotin and provides an amplified flour that emits light when the chip is then scanned with a confocal laser and the distribution pattern of signal in the array is recorded (Ref 53)) A scanner analyzes the GeneChip for signals. Advanced algorithms are then used to give data on the expression levels of the genes of interest.

For a detailed explanation of how the entire process of using Affymetrix GeneChips, including RNA extraction and amplification, is currently being implemented in laboratories see Gene Expression Studies.

Other Variations

One great advantage of microarrays is their flexibility. Many different platforms exist, and many more can be created. The two platforms outlined above may be modified as needed by a researcher. For example if a particular experiment requires the use of DNA as a target instead of RNA it could be easily implemented.

For clinical use, the most important microarray to date is the Roche AmpliChip CYP450. The AmpliChip CYP450 is “world's first pharmacogenomic (See Pharmacogenomics) microarray designed for clinical applications.” (Ref. 52)The chip is based on Affymetrix GeneChip technology, but is designed specifically for clinical use.

Other commercial variations are also found and include Nanogen's NanoChip.

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). These 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

Techniques of molecular biology

Since the late 1950s and early 1960s, molecular biologists have learned to characterize, isolate, and manipulate the molecular components of cells and organisms. These components include DNA, the repository of genetic information; RNA, a close relative of DNA whose functions range from serving as a temporary working copy of DNA to actual structural and enzymatic functions as well as a functional and structural part of the translational apparatus; and proteins, the major structural and enzymatic type of molecule in cells.

Macromolecule blotting and probing

The terms northern, western and eastern blotting are derived from what initially was a molecular biology joke that played on the term Southern blotting, after the technique described by Edwin Southern for the hybridisation of blotted DNA. Patricia Thomas, developer of the RNA blot which then became known as the northern blot actually didn't use the term. Further combinations of these techniques produced such terms as southwesterns (protein-DNA hybridizations), northwesterns (to detect protein-RNA interactions) and farwesterns (protein-protein interactions), all of which are presently found in the literature.

Polymerase chain reaction

The polymerase chain reaction is an extremely versatile technique for copying DNA. In brief, PCR allows a single DNA sequence to be copied (millions of times), or altered in predetermined ways. For example, PCR can be used to introduce restriction enzyme sites, or to mutate (change) particular bases of DNA, the latter is a method referred to as "Quick change". PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library. PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and, more recently, real-time PCR (QPCR) which allow for quantitative measurement of DNA or RNA molecules.