Definition: An assay is an analysis done to determine the presence of a substance, its potency, and the amount of a substance.
Assays are tests that use biomarkers (indicators) to determine the presence, and measure the amount and activity of a substance. A test that measures the toxicity or effect of a given substance on an organism’s cell is a bioassay. An immunoassay determines how much, if any, of the substance is present inside the organism. In immunoassays, an antibody or antigen is added to the substance or an enzyme and its substrate. A specific antibody molecule will bind exclusively with its antigen. For example, ELISA (enzyme-linked immunosorbent assay) is an assay that uses color change as a biomarker to test for and estimate the concentration of antibodies and antigens or enzyme and substrates. Automated assays use spectrometry (the measuring different emissions and absorption of electromagnetic radiation to identify a substance). ELFA is a type of ELISA (enzyme-linked fluorescent assay), where instead of a color change, a computer uses a laser to emit a wavelength of electromagnetic radiation (light) on the substance, and if the antibody and antigen or substrate and enzyme combine, then they will emit a signature wavelength of electromagnetic radiation and fluoresce (glow) and the intensity of the glow can be measured by the computer to calculate the concentration of the substance. In addition, fluorescent biomarkers can be added to the genomes of organisms to produce proteins in the cell that will bind to specific sites within the cell or organism and fluoresce under certain light.
Biological Assays Tutorials:
NCBI
The University of Arizona
VectorLabs ®
Bio-Rad ®
To isolate DNA from a cell in solution, the cell membrane and in some cases, the cell wall, needs to be lysed (broken open). The phospholipids bilayer membrane is washed away with a detergent and proteins are broken down with enzymes. DNA is precipitated out of the solution the DNA by adding cold ethanol or isopropanol. The precipitated DNA is placed in a centrifuge and aggregates at the bottom.
DNA extraction tutorial:
University of Utah
Carleton College
Definition: DNA Fingerprinting is the identification of the DNA of different individuals based on variation that exists between them in the sequence of bases in the DNA.
DNA typing involves identifying or comparing identifying markers on fragments of DNA to create a profile of an individual that can be used to identify genetic relations or for forensic purposes. These markers are sequences of nucleotides. The sequences can be located using RFLP analysis (restriction fragment length polymorphisms) of gel electrophoresis size bands, or using PCR analysis of AmpFLP (amplified fragment length polymorphisms) or Single Nucleotide Polymorphisms (SNPs), which focus on short tandem nucleotide repeats (STRs) or variable number tandem nucleotide repeats (VNTRs). These polymorphisms and repeat markers are simply nucleotide sequences on fragments of DNA that are similar among closely related individuals, but are highly variable among non-related individuals. The data from forensic genetic typing in the US is collected in a database called CODIS. According to the FBI website, The National DNA Index (NDIS) within CODIS contains over 8,483,906 offender profiles as of June 2010.
DNA Fingerprinting Tutorials:
NCBI's SNPs
NCBI's Gene Mapping
PBS NOVA
Universidad de Pais Vasco
Definition: Epigenetics is a suite of technologies used to interrupt or suppress the expression of a gene at the transcriptional or translational levels.
Gene silencing purposefully causes mutations in a gene by deleting it or disrupting its expression within cells. Scientists gain information about the role of the gene and its protein products by silencing it and observing the resulting consequences in an organism’s development or cellular function. If the cell’s RNA polymerase (an enzyme that copies DNA during transcription) is prevented from copying the DNA sequence of nucleotides on a given gene, there would be no mRNA created to build a protein (no translation). By “switching off” this gene, its protein will not be made. Scientists then observe the developmental and cellular consequences of the protein’s absence.
Epigenetics Gene silencing technologies such as transvection (disrupting the mitotic or meiotic pairing of homologous chromosomes that activates or suppresses an allele on a gene) or VIGS (viral-induced RNA gene silencing) or RNAi (RNA interference) suppressing gene expression by introducing RNA into the cell to disrupt mRNA (messenger RNA) and prevent it from being translated into a protein. Epigenetics is the altering of the phenotype (gene expression) and not the genotype (the actual DNA code or sequence). In other words, epigenetics interferes with the way the cell regulates the expression of a gene and does not mutate the gene as in causing methylation (a chemical process that suppresses gene activity). Whereas mutations such as deletions, additions, repeats or inversions alter the gene’s DNA nucleotide sequence and disrupts or ceases its successful expression.
Gene Silencing Tutorials:
NCBI
PBS NOVA
Cold Spring Harbor Laboratory Epigenetics Tutorial (chapter 29-41)
University of Maryland
For more information:
Waterhouse and Helliwell's article on the utilization of RNAi on plant genomes.
Cambridge University
Cold Spring Harbor Laboratory
Journal of RNAi and Gene Silencing
Definition: Gel Electrophoresis is a technique where molecules (DNA, proteins, etc.) migrate through a gel matrix in response to an applied electrical current. Gel electrophoresis allows molecules to be separated on the basis of size, electric charge and other physical properties.
Gel Electrophoresis is a technique used to gauge the size of molecules like fragments of DNA and used for preparations of Southern blots (see in index). The process involves using a tray filled with an agarose or polyacrylamide gel and buffer solution that has electric current running through it. The samples are stained for visibility and placed in wells at one end of the tray using a pipette. Since smaller molecules usually diffuse faster through the matrix than larger ones, the bands of fragments that diffuse closer to the end are considered larger. In addition to the molecules in question, substances of known size are added to some of the wells to be used as markers. DNA and RNA are measured in kilobases (kb), which is a unit of measurement equal to 1000 base pairs (nucleotides). The bands of fragments can be viewed under UV light and photographed.
Southern Blot is a method for identifying a specific sequence of DNA using a hybridization probe (complimentary Base pairs of DNA or RNA) used before automation. It involves using a cellulose membrane onto which the DNA is transferred from the gel. The membrane is heated or exposed to ultraviolet radiation to attach the transferred DNA to the membrane. The hybridization probe contains a biomarker that will change color or fluoresce when if it finds the matching sequence. If the probe matches a specific sequence in the sample, the probe will attach itself and activate the biomarker. After hybridization, it can be photographed or fluorescence can be scanned.
In automated DNA sequencing, the large amounts of fragments (amplified in vivo/in vitro) that would have been added to the gel are loaded into a DNA sequencer, which is a machine that uses lasers to “read” the nucleotides. When exposed to the laser, each nucleotide emits a specific wavelength that is recorded by the computer as a specific color (T=red, A=green, C=blue, and G=yellow). Instead of a black and white photograph of bands representing each nucleotide sorted by size taken under UV light after electrophoresis, the automated DNA sequencer produces an electropherogram, which is a read-out of each nucleotide represented by peaks of different colors. The larger the amount of DNA sample, the darker the peaks.
Gel Electrophoresis tutorials:
Rochester Institute of Technology
University of Utah
University of Illinois
Colorado State University
Automated DNA sequencing
U.S. Department of Energy
For more information:
BioQUEST Library Module on Southern Blotting (University of Wisconsin and Michigan State University)
American Electrophoresis Society
Wiley Books and Journals on Electrophoresis and separation sciences
Definition: Genetic engineering is the process of manually adding new DNA on a molecular level with the goal of adding one or more new traits that are not already found in that organism. Genetic engineering includes recombinant DNA and gene splicing technologies.
Recombinant DNA is a form of transgenic technology that involves adding a gene from one organism (exogenous DNA) into the genome of another. For example, the bacterium E. coli can produce human insulin. The E. coli’s plasmid (circular DNA molecule found in a bacterium) is removed. Using restriction enzymes and ligases like scissors and glue, a human insulin-producing gene can be spliced into the plasmid and reintroduced into the bacterium. During transcription and translation, the bacterium’s cellular machinery will produce the human recombinant protein insulin.
The process of transformation is a type of recombinant technology where the cell takes up and expresses exogenous DNA. For example, bacteria can take up genes they encounter in their environment or by conjugating with other bacteria and exchanging genes. The exogenous DNA is incorporated into its plasmid and begins to express the gene. This explains how bacteria that are resistant to certain antibiotics can “share” this genetic ability.
Other ways of introducing genes into a genome are transduction and transvection. In transduction, the exogenous DNA is carried into a cell by a carrier virus. For instance, a bacteriophage is a virus that can infect bacteria and carry exogenous DNA that will be spliced into the plasmid or genome by the virus. Transfection refers to the introduction of exogenous nucleic acids into the cell through non-viral means such as diffusion across a cell’s membrane.
Genetic Engineering Tutorials:
Recombinant DNA tutorial
Cold Spring Harbor Laboratory
US Department of Energy
Rensselaer Polytechnic Institute
Definition: Sometimes called a gene chip or a DNA chip, microarrays consist of large numbers of molecules (often, but not always, DNA) distributed in rows in a very small space. Microarrays permit scientists to study gene expression by providing a snapshot of all the genes that are active in a cell at a particular time.
Similar to ELFA assays, microarrays are a kind of test that can measure gene activity or the effect of a substance on gene activity by using fluorescence. The technology involves using a small glass or plastic slide that contains microscopic samples of an organism’s genes (probes or reporters). During gene expression (protein synthesis), mRNA is active. If a gene is not expressed, the mRNA is not active in the transcription and the translation of that gene (building the protein). A control (healthy) mRNA sample and an experimental (diseased or targeted) mRNA are tagged with different color fluorescing biomarkers. When the mRNA is added to the chip, the active genes will fluoresce and the resulting fluorescence can be measured.
Microarrays Tutorials:
NCBI
Cold Spring Harbor Laboratory
Oregon State University
Bioinformatics Tutorials
Public Library of Science
Definition: polymerase chain reaction (PCR): A laboratory technique for rapidly synthesizing large quantities of a specific DNA segment. The procedure involves separating the DNA into its two complementary strands, using DNA polymerase to synthesize two-stranded DNA from each single strand, and repeating the process until a massive number of identical DNA particles have been synthesized.
PCR stands for polymerase chain reaction, and is a laboratory technique used to speed up the process of DNA replication or amplification (exponential copying from just a few strands) of DNA. A PCR machine is called a thermal cycler, and uses heat to separate the double helical and complementary strands of DNA. Each separated single strand is called a template. An enzyme (a catalyst that speeds up a reaction) called DNA polymerase, and nucleotides (the building blocks of DNA Adenine, Thymine, Cytosine and Guanine) are added to the reaction. Fragments are created using restriction enzymes which act like scissors and can cut DNA sequences at specific points. The polymerase, the enzyme involved in DNA replication, copies each template by assembling nucleotides, and using primers, which are the short DNA fragments that attach to the template, and then targets and copies multiple areas of the template simultaneously. The term chain reaction refers to the repeated and exponential copying of DNA in that each new DNA molecule newly produced is then subsequently separated and copied, creating a great number of copies in a short period of time.
PCR Tutorials:
University of Utah Virtual PCR Lab
Cold Spring Harbor Laboratory
Cold Spring Harbor's PCR animation tutorial
Cold Spring Harbor's DNA Manipulation tutorial
More information:
Scientific Journal: Biotechniques: International Journal of Life Science Methods Archives
Somatic Cell Nuclear Transfer Cloning refers to the cloning of an organism by transferring the nucleus from the somatic cell (non-gamete or not a sperm or an ovum) of the organism that is to be cloned into a zygote (donor cell) that has had its nucleus removed, and then is implanted into the uterus of a donor mother. The resulting offspring will be an identical copy of the organism that supplied the somatic cell nucleus.
Cloning Tutorials:
University of Utah
Human Genome Project
Howard Hughes Medical Institute
Definition: A stem cell is an undifferentiated cell whose daughter cells may differentiate into other cell types (such as blood cells)
Stem cells are undifferentiated cells. There are many types of stem cells found in a variety of tissues. Stem cells are characterized by their ability to continually to divide without differentiating into a specific cell type (self-renewal) and by having the potential ability to differentiate into a variety of cell types (pluripotency). The two major types of stem cells are embryonic and adult stem cells. Embryonic stem cells are an organism’s initial cells following fertilization. This microscopic collection of cells is called a blastocyst and its cells are totipotent and pluripotent (they have the ability to become a full organism as well as any type of cell in the body). Adult stem cells (also called somatic stem cells) are found naturally in varying numbers in different tissues and have limited potency but can be reprogrammed by activating embryonic genes to become like embryonic stem cells (induced pluripotent stem cells, iPSCs).
Stem Cell Tutorials:
NIH
University of Massachusetts International Stem Cell Registry
Howard Hughes Medical Institute
Harvard HHMI/MCB
For more information:
The NIH provides information on stem cell culturing and scientific literature on stem cells.
The International Stem Cell Registry at the University of Massachusetts, which includes literature on specific culturing properties for various stem cell lines.
A database of research articles that feature human embryonic stem cell lines that are currently listed in the registry.
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