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Tutor profile: Rhea A.

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Rhea A.
Graduate Teaching Assistant at Virginia Commonwealth University
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Subject: Chemistry

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Question:

You are developing a rapid Gas Chromatography assay for the detection/quantification of amino acids and aromatic hydrocarbons within the animal feed. Since you are looking for a limited number of specific compounds, you can use non-mass spectrometry-based detectors for the back-end of your GC setup. Among all of the possible detectors for GC that are not mass spectrometry, which would you choose for detection of amino acids, and which would you choose for detection of aromatic hydrocarbons?

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Rhea A.
Answer:

Gas chromatography (GC) separates and detects molecules using a carrier gas. The sample can be gas or liquid. For the liquid sample, it needs to be first vaporized and then separated. The analyte interacts with the detector when it gets eluted from the column. This interaction is converted into an electronic signal by the detector, which is then supplied to the data system for the estimation of molecules. There are many detectors that are used in combination with GC. Some detectors can detect any analyte while other detectors detect only specific type of analyte. Thermal Conductivity Detector (TCD) is the universal detector that can detect hydrogen, nitrogen and several other compounds. This detector helps in detecting the changes in temperature or thermal conductivity. It has two tubes that contain gas and heating coils. There is a carrier gas like Helium that flows through one of the tubes and the sample to be tested passes through the other tube. When the sample comes in contact with the carrier gas (Helium), there is a change in the thermal conductivity. This change in the thermal conductivity produces a signal which is proportional to the concentration of the sample molecules. TCD is a non-specific and universal detector. It can help in the detection of aromatic hydrocarbons but the sensitivity of this detector is lower than the sensitivity of the flame ionization detector (FID). FID is more suitable for the detection of aromatic hydrocarbons. FID detector is more specific for compounds with carbon-hydrogen bonds and since aromatic hydrocarbons have C-H bonds, it provides good resolution. Flame ionization generates organic ions. When the sample contains organic molecules, the FID has a flame that combusts these organic molecules into organic ions. It breaks down the molecules which are then detected at the electrode which produces a change in current. In this detector, the change in current is measured; the current change would help in estimating the number of molecules. The sample gets introduced into the flame and the flame breaks organic molecules into constituent species which are positively charged. These species then hit the electrode which produces a change in current. In FID, a hydrogen-air flame is used to ionize the sample. Once the ions are formed, the detector collects the ions and produces a current, and this current is then converted into an electrical signal. The limit of detection of FID is in the low picogram range. There is another type of detector called the Photoionization detector (PID) that is used to analyze aromatic hydrocarbons. This detector uses ultraviolet light to ionize the sample and the resulting ions produced by this process are collected at the electrode. A current is generated which thereby measures the concentration of the sample. The sensitivity of this detector lies in the picogram range. For the detection of amino acids, Nitrogen-Phosphorus Detector (NPD) can be used. Amino acids make up proteins. They consist of an amino group that contains nitrogen (NH2) and a carboxyl group. NPD detector is specific to nitrogen or phosphorus-containing compounds and since amino acids have nitrogen, they can be easily detected by NPD. The key component in NPD is the Rubidium beads. The compounds exiting the column collide with the rubidium ions and undergo a chemical reaction. The rubidium bead is heated by the flame and due to the high temperature, rubidium ions are formed which interact with the nitrogen or phosphorus-containing compounds. The resulting ions are attracted towards the electrode and a change in current is produced which is then detected and quantified. The sensitivity of this detector lies in the range of 1-10pg.

Subject: Biochemistry

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Question:

Promega’s Plexor HY system is a quantitative PCR assay with a methodology different from the SYBR Green method of qPCR. Compare and contrast both the methods.

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Rhea A.
Answer:

Real-time quantitative PCR, or qPCR is a standard method for detecting and quantifying DNA in the sample. One of the most basic and common qPCR procedures is to utilize fluorescent DNA-binding dyes like SYBR Green. This dye is added to the reaction and the fluorescence is evaluated after each cycle. Because the fluorescence of the dye increases considerably when double-stranded DNA is present, DNA synthesis can be tracked as a rise in fluorescent signal. The SYBR green method of qPCR involves the quantification of human DNA. Promega’s Plexor HY system is a quantitative PCR assay that determines male and total human DNA concentrations simultaneously. The steps involved in this qPCR assay are- 1. Primer extension and annealing 2. Incorporation of dabcyl-iso-dG 3. Quenching The Plexor® HY system results in a reduction in signal as the product accumulates because of its quenching mechanism. Plexor® HY, unlike other qPCR assays that use probes or intercalating dyes, relies on quenching between two nucleotides, isoguanine (iso-dG) and 5'-methylisocytosine (iso-dC). The amplification process requires basically the use of two primers. The assay involves the use of a fluorescently labeled iso-dC on the 5' end of one of the two primers. The quencher-containing iso-dG is precisely integrated at a place complementary to the iso-dC in the opposite strand during the amplification process. Iso-dG is modified with the quencher dabcyl. When the quenching takes place, there is a fluorescent signal reduction which is in proportion to the amount of dsDNA present. This allows quantification during amplification. An internal PCR control is included in the kit to check for false-negative findings that may arise when PCR inhibitors are present. This kit detects DNA down to 6.4pg. It enables the determination of the melting temperature (Tm) of the products following amplification using a melt curve or dissociation curve. This is useful for determining the reaction's specificity. The Plexor® HY System's fluorescein dye is utilized to detect a human autosomal DNA target. On chromosome 17, the primers amplify a multicopy, 99bp target. This reaction's results are used to calculate the total amount of human DNA in a sample. The Plexor® HY System's CAL Fluor® Orange 560 dye is utilized to detect a Y-chromosomal DNA target. On the Y chromosome, the primers amplify a multicopy 133bp target. The total amount of human male DNA in a sample is calculated using the results of this reaction. The amplification plot for this assay is different from the qPCR which involves the use of SYBR green as an intercalating dye. SYBR Green is a nonspecifically intercalating dsDNA-binding dye that allows the amount of PCR product to be measured. As the amplification process progresses, the amount of DNA product produced rises, and the number of SYBR green molecules incorporated into DNA increases as well. SYBR Green qPCR can be used for relative DNA quantification because the rise in fluorescence is proportional to the amount of product accumulated. The data gathered during the exponential phase of the reaction provides quantitative information about the amplification target's starting quantity. The result of SYBR Green analysis is a rise in fluorescence intensity proportionate to the amount of amplicon produced. An amplification curve depicting the presence of amplified product as fluorescent signal vs cycle number is the major result of this real-time qPCR. From the curve, the quantification of the sample can be done. The quantification cycle (Cq or Ct) is used to calculate the amount of starting material. The amount of starting template in the reaction is inversely related to Cq or Ct value.

Subject: Biology

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Question:

How might Bryophytes and Lycophytes have contributed to the evolution of flowering plants and other land plants?

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Rhea A.
Answer:

Plants are divided into two major categories: non-vascular and vascular plants. Both groups include embryos that are multicellular, suggesting that they are strongly linked with one another. Bryophytes are the plants that are non-vascular and present on land and lycophytes are the land plants that are vascular. All other land plants such as ferns, gymnosperm, and angiosperm come under the category of vascular land plants. ●Plants have developed a special form of leaf structure called microphyll around 380 million years ago, and some plants have developed megaphylls around 350 million years ago. Microphylls are found in lycophytes. Angiosperm and gymnosperm leaves are derived from simple microphylls and megaphylls that demonstrate the evolution of flowering plants and other land plants. ●Lycophytes act as a base for all other vascular plants. While they resemble bryophytes superficially, they contain vascular tissues that are not present in the bryophytes. The reproductive cycle of lycophytes is similar to other vascular plants as well. The bryophytes have the same life cycle as that of gymnosperms and angiosperms. It is defined by the alternation of generations and this shows an evolutionary link between the plants. ●Many adaptations have evolved from bryophytes for life on land, such as adaptations in the reproductive system. For example, the offspring develop from embryos that are multicellular and stay connected to the plant. The plant provides protection and nourishment to the embryos. Bryophytes also had a structure called gametangia that provided protection against the drying of plant gametes which was a significant adaptation that interlinks them with the vascular land plants. ●The evolution of spores The spores have evolved into seed ferns. These seed ferns started developing female gametes which remained with the parent plant and waited for the arrival of the male gamete. Slowly, these seedless ferns gave rise to seed plants. ●First to inhabit the land Bryophytes (amphibians) are the first land plants. The alteration of the generation in this phylum is clearly demarcated. The gametophyte is independent of the sporophyte in these organisms, however, these are the plants that are the connecting link between aquatic plants and the exclusively terrestrial plants. ●Development of vascular tissue/system The huge land trees are supported through the vascular system present in them. The bryophytes were the first plants to have developed the primitive forms of vascular tissues. Algae lacked such a system to support the growth of the organisms. The evolution of different kinds of vascular tissues supported the growth of the plants on the land. Lycophytes are considered the oldest land plants to have a vascular system. They existed or evolved before flowering plants or any other land plants. ●Dominant sporophyte in lycophytes Lycophytes show an independent sporophyte, unlike bryophytes and algae. The independence of sporophyte is found in higher plant phyla like Gymnosperms and Angiosperms. The bryophytes and algae, both have a dominant gametophytic phase. The evolution of a diploid independent sporophyte is seen in lycophytes. ●Cuticle layer in bryophytes Bryophytes lack the ability to regulate water loss, however, many of them have cuticle layers just like higher plants. The cuticle layer (waterproof) can prevent water loss. ●Root like structure (rhizoids) in bryophytes and root development in lycophytes There are root-like structures present in some bryophytes that help absorb water from the substratum. Root development has been one of the crucial characteristics in the evolution of terrestrial plants or vascular plants. The conducting tissue like the xylem provided the way of absorbing water from the soil and also to provide it to organs of the plant.

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