Genetic testing allows the genetic diagnosis of vulnerabilities to inherited diseases, and can also be used to determine a person's ancestry. Normally, every person carries two copies of every gene, one inherited from their mother, one inherited from their father. The human genome is believed to contain around 20,000 - 25,000 genes. In addition to studying chromosomes to the level of individual genes, genetic testing in a broader sense includes biochemical tests for the possible presence of genetic diseases, or mutant forms of genes associated with increased risk of developing genetic disorders. Genetic testing identifies changes in chromosomes, genes, or proteins. Most of the time, testing is used to find changes that are associated with inherited disorders. The results of a genetic test can confirm or rule out a suspected genetic condition or help determine a person's chance developing or passing on a genetic disorder. Several hundred genetic tests are currently in use, and more are being developed.
Histones are subject to a wide variety of posttranslational modifications including but not limited to, lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation (Vasquero 2003). These modifications occur primarily within the histone amino-terminal tails protruding from the surface of the nucleosome as well as on the globular core region (Cosgrove 2004).
Histone modifications are proposed to affect chromosome function through at least two distinct mechanisms. The first mechanism suggests modifications may alter the electrostatic charge of the histone resulting in a structural change in histones or their binding to DNA. The second mechanism proposes that these modifications are binding sites for protein recognition modules, such as the bromodomains or chromodomains, that recognize acetylated lysines or methylated lysine, respectively.
The existence of these modifications and recognition modules led to a well established “histone code” hypothesis proposed by Strahl and Allis (2000). Overall, posttranslational modifications of histones create an epigenetic mechanism for the regulation of a variety of normal and disease-related processes.
Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization. The method is named after its inventor, the British biologist Edwin Southern.The southern blot is used to verify the presence or absence of a specific nucleotide sequence in the DNA from different sources and to identify the size of the restriction fragment that contains the sequence.
In this procedure, the DNA is isolated from each source and then digested with a specific restriction enzyme. The DNA restriction fragments are then loaded onto an agrose gel and the fragments separated by electrophoresis according to size, with the smaller fragments migrating faster than larger fragments. The DNA is then transferred from the fragile gel to a nylon filter.
Next the radioactively labeled nucleic acid probe is added. The probe binds to complementary DNA segments. Note that the DNA segment being probed is not present in organism B
To detect the position of the radioactive probe, the nylon membrane is covered with an X-ray film. After development, the positions of the probe become visible.
Gene expression is the process by which the inheritable information in a gene, such as the DNA sequence, is made into a functional gene product, such as protein or RNA.
Several steps in the gene expression process may be modulated, including the transcription step and the post-translational modification of a protein. Gene regulation gives the cell control over structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism. Gene regulation may also serve as a substrate for evolutionary change, since control of the timing, location, and amount of gene expression can have a profound effect on the functions (actions) of the gene in the organism.
Non-protein coding genes (e.g. rRNA genes, tRNA genes) are not translated into proteins.
Measurement
The expression of many genes is regulated after transcription (i.e., by microRNAs or ubiquitin ligases), so an increase in mRNA concentration need not always increase expression. Nevertheless, mRNA levels can be quantitatively measured by Northern blotting, a process in which a sample of RNA is separated on an agarose gel and hybridized to a radio-labeled RNA probe that is complementary to the target sequence. Northern blotting requires the use of radioactive reagents and can have lower data quality than more modern methods (due to the fact that quantification is done by measuring band strength in an image of a gel), but it is still often used. It does, for example, offer the benefit of allowing the discrimination of alternately spliced transcripts.
A more modern low-throughput approach for measuring mRNA abundance is real-time polymerase chain reaction or RT-PCR. With a carefully constructed standard curve RT-PCR can produce an absolute measurement such as number of copies of mRNA per nanolitre of homogenized tissue. The lower level of noise in data obtained via RT-PCR often makes this the method of choice, but the price of the required equipment and reagents can be prohibitive.
In addition to low-throughput methods, transcript levels for many genes at once (expression profiling) can be measured with DNA microarray technology or "tag based" technologies like Serial analysis of gene expression (SAGE) or the more advanced version SuperSAGE, which can provide a relative measure of the cellular concentration of different messenger RNAs. Recent advances in microarray technology allow for the quantification, on a single array, of transcript levels for every known gene in the human genome. The great advantage of tag-based methods is the "open architecture", allowing for the exact measurement of any transcript, known or unknown. Especially SuperSAGE recommends itself therefore also for studying organisms with unknown genomes.
Protein levels themselves can be estimated by a number of means. The most commonly used method is to perform a Western blot against the protein of interest, whereby cellular lysate is separated on a polyacrylamide gel and then probed with an antibody to the protein of interest. The antibody can either be conjugated to a fluorophore or to horseradish peroxidase for imaging or quantification. Another commonly used method for assaying the amount of a particular protein in a cell is to fuse a copy of the protein to a reporter gene such as Green fluorescent protein, which can be directly imaged using a fluorescent microscope. Because it is very difficult to clone a GFP-fused protein into its native location in the genome, however, this method often cannot be used to measure endogenous regulatory mechanisms (GFP-fusions are therefore most often expressed on extra-genomic DNA such as an expression vector). Fusing a target protein to a reporter can also change the protein's behavior, including its cellular localization and expression level.
The pattern of detection of a gene or gene product may be described using terms such as facultative, constitutive, circadian, cyclic, housekeeping, or inducible.
Regulation of gene expression
Regulation of gene expression is the cellular control of the amount and timing of appearance of the functional product of a gene. Any step of gene expression may be modulated, from the DNA-RNA transcription step to post-translational modification of a protein. Gene regulation gives the cell control over structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism.
Expression system
An expression system consists, minimally, of a source of DNA and the molecular machinery required to transcribe the DNA into mRNA and translate the mRNA into protein using the nutrients and fuel provided. In the broadest sense, this includes every living cell capable of producing protein from DNA. However, an expression system more specifically refers to a laboratory tool, often artificial in some manner, used for assembling the product of a specific gene or genes. It is defined as the "combination of an expression vector, its cloned DNA, and the host for the vector that provide a context to allow foreign gene function in a host cell, that is, produce proteins at a high level".
In addition to these biological tools, certain naturally observed configurations of DNA (genes, promoters, enhancers, repressors) and the associated machinery itself are referred to as an expression system, as in the simple repressor 'switch' expression system in Lambda phage. It is these natural expression systems that inspire artificial expression systems, (such as the Tet-on and Tet-off expression systems).
Each expression system has distinct advantages and liabilities, and may be named after the host, the DNA source or the delivery mechanism for the genetic material. For example, common expression systems include bacteria (such as E.coli), yeast (such as S.cerevisiae), plasmid, artificial chromosomes, phage (such as lambda), cell lines, or virus (such as baculovirus, retrovirus, adenovirus).
Overexpression
In the laboratory, the protein encoded by a gene is sometimes expressed in increased quantity. This can come about by increasing the number of copies of the gene or increasing the binding strength of the promoter region.
Often, the DNA sequence for a protein of interest will be cloned or subcloned into a plasmid containing the lac promoter, which is then transformed into the bacterium Escherichia coli. Addition of IPTG (a lactose analog) causes the bacteria to express the protein of interest. However, this strategy does not always yield functional protein, in which case, other organisms or tissue cultures may be more effective. As for example the yeast, Saccharomyces cerevisiae, is often preferred to bacteria for proteins that undergo extensive Posttranslational modification. Nonetheless, bacterial expression has the advantage of easily producing large amounts of protein, which is required for X-ray crystallography or nuclear magnetic resonance experiments for structure determination.
Gene networks and expression
Genes have sometimes been regarded as nodes in a network, with inputs being proteins such as transcription factors, and outputs being the level of gene expression. The node itself performs a function, and the operation of these functions have been interpreted as performing a kind of information processing within cell and determine cellular behaviour.
Northern blot is a technique used in molecular biology research to study gene expression. It takes its name from its similarity to the Southern blot technique, named for biologist Edwin Southern. The major difference is that RNA, rather than DNA, is analyzed in the northern blot. Both techniques use electrophoresis and detection with a hybridization probe. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.
A northern blot is very similar to a Southern blot except that it is RNA rather than DNA which is extracted, run on a gel and transferred to a filter membrane. There are 3 types of RNA: tRNA (transfer RNA - active in assembly of polypeptide chains), rRNA (ribosomal RNA - part of the structure of ribosomes) and mRNA (messenger RNA - the product of DNA transcription and used for translation of a gene into a protein). It is mRNA which is isolated and hybridized in northern blots.
mRNA is extracted from the cells grown in galactose and cells grown in glucodse and purified.
The mRNA is loaded onto a gel for electrophoresis. Lane 1 has gal mRNa Lane 2 has the Glucose mRNA.
An electric current is passed through the gel and the RNA moves away from the negative electrode. The distance moved depends on the size of the RNA fragment. Since genes are different sizes the size of the mRNAs varies also. This results in a smear on a gel. Standards are used to quantitate the size. The RNA can be visualized by staining first with a fluorescent dye and then lighting with UV.
RNA is single-stranded, so it can be transferred out of the gel and onto a membrane without any further treatment. The transfer can be done electrically or by capillary action with a high salt solution.
A GAL DNA probe is incubated with the blot.the single stranded GAL DNA probe binds with immobilized GAL mRNA The blot is washed to remove non-specifically bount probe and then a development step allows visualization of the probe that is bound.
