Question

Please explain the processes, best uses, advantages and notable details in regards to  DNA sequencing, Promoter Assay using Reporter Molecules, Real-Time PCR, Array Analysis of RNA, Western Blots and Immunodetection.

Answer 1

DNA sequencing is the process of determining the sequence of nucleotides within a DNA molecule. Every organism’s DNA consists of a unique sequence of nucleotides. Determining the sequence can help scientists compare DNA between organisms, which can help show how the organisms are related. This means that by sequencing a stretch of DNA, it will be possible to know the order in which the four nucleotide bases – adenine, guanine, cytosine, and thymine – occur within that nucleic acid molecule.

steps:- Sample preparation (DNA extraction), PCR amplification of target sequence, Amplicons purification, Sequencing pre-prep, DNA Sequencing, Data analysis.

• in DNA sequensing sample tube placed into the DNA sequencer. Here first, the PCR product (which is now our sample for sequencing) is denatured into the heat govern step and becomes single-stranded. The high fidelity Taq DNA polymerase adds the labelled nucleotide to the growing DNA strand. That signals of the addition of each complementary nucleotides are recorded by the machine and the data is sent to the computer.

ADVANTAGE:- Knowledge of DNA sequences has become indispensable for basic biological research, and in numerous applied fields such as medical diagnosis, biotechnology, forensic biology, virology and biological systematics. Comparing healthy and mutated DNA sequences can diagnose different diseases including various cancers,characterize antibody repertoire, and can be used to guide patient treatment.Having a quick way to sequence DNA allows for faster and more individualized medical care to be administered, and for more organisms to be identified and cataloged.

Promoter Assay using Reporter Molecules: Reporter genes can be used to assay for the activity of a particular promoter in a cell or organism.In this case there is no separate "gene of interest"; the reporter gene is simply placed under the control of the target promoter and the reporter gene product's activity is quantitatively measured. The results are normally reported relative to the activity under a "consensus" promoter known to induce strong gene expression.

• A more complex use of reporter genes on a large scale is in two-hybrid screening, which aims to identify proteins that natively interact with one another in vivo.

REAL TIME PCR:- is a technique used to monitor the progress of a PCR reaction in real time. At the same time, a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified.  In a real time PCR protocol, a fluorescent reporter molecule is used to monitor the PCR as it progresses. The fluorescence emitted by the reporter molecule manifolds as the PCR product accumulates with each cycle of amplification.

Array Analysis of RNA:- have become a powerful approach for comparing complex sample RNA populations. Using array analysis, the expression profiles of normal and tumor tissues, treated and untreated cell cultures, developmental stages of an organism or tissue, and different tissues can be compared. A typical gene array experiment involves:

1. Isolating RNA from the samples to be compared
2. Converting the RNA samples to labeled cDNA via reverse transcription; this step may be combined with aRNA amplification
3. Hybridizing the labeled cDNA to identical membrane or glass slide arrays
4. Removing the unhybridized cDNA
5. Detecting and quantitating the hybridized cDNA, and
6. Comparing the quantitative data from the various samples

Western Blots and Immunodetection:-

Immunodetection includes any assay technique used to detect and quantify amino acids, antibodies, and hormones. The most common forms are ELISA and Western Blots. Make sure that you have as clean samples as possible before you start using these methods as sample purity greatly affects the downstream outcome.

• Western Blot is immunodetection technique used for detection of proteins. Blotting refers to the process of moving biological samples from a gel to a membrane, with detection steps being performed on the surface of the membrane. The chromatographic properties of PAGE electrophoresis are used to separate macromolecules, which are then transferred (i.e. blotted) to a membrane, usually through physical and electrical methods. Next, the membrane is probed with antibodies specific to your molecule of choice. Secondary antibodies, which are bound to a detector enzyme, are then added. Following the same procedure as ELISA, the enzyme's substrates will change the color of the membrane in the presence of your target molecule. Otherwise the protein can be transferred to a photographic film and visualized by chemiluminescence via the reporter on the secondar antibody