cnwokemodoih's blog

DNA extraction is only the first step in exploring the genes that exist in the genome. When we finally have the genomic DNA of an organism, we need to know how much DNA we have and how pure the DNA is. We'll need to evaluate the quality and quantity of our DNA. The quality of our DNA can be examined by using Nanodrop. This enables us to see how purity (A260/280) and concentration of our DNA. This knowledge will influence how DNA we use in subsequent processes like PCR, in-vitro transcription and so on. The ideal purity of DNA is 1.8. Newly extracted DNA is likely to have its A260/280 exceed 1.8, indicating the presence of impurities like RNA. We can visualize our DNA by using gel electrophoresis. We pour agarose gel, containing ethidium bromide, into a rig with a comb. We add specific volumes of our samples and a ladder to the wells formed in the gel by the comb. The ladder is needed as a yardstick against which we can deduce the approximate length of strands. Seeing as DNA is negative, it runs towards the positive end of the gel and add varying speeds depending on the size of a strand. The final gel can be viewed under UV light to visualize the distinct bands. 

Dravet's syndrome is a rare, severe, genetic epileptic encephalopathy. Most cases of Dravet's syndrome are caused by a mutation in the SCN1A gene. This gene codes for a sodium channel, voltage gated type 1 alpha subunit. Most of these mutated genes are expressed in GABAergic neurons; hence, GABAergic neurotransmission is compromised. GABA is a neurotransmitter with vast inhibitory functions. As such, seizurelike locomotor behavior and epileptiform brain activity are signs of Dravet's syndrome. In humans, the presence of one mutant allele is sufficient enough for the display of the mutant phenotype but in zebrafish, the common model organism for studying neural networks and disorders, the mutant phenotype is only observed in larvae that have two copies of the mutant allele. Though GABAergic neurotransmission has been found to be most directly affected by the mutations underlying Dravet's syndrome, scientists have explored the potential of other neurotransmitters rescuing the phenotype, relieving mutant zebrafish larvae of epileptic seizures. One of the neurotransmitters that has shown some promise is serotonin (5-hydroxytryptamine). It's been revealed that increasing serotonin levels can reduce epileptic locomotor and brain activity. The drug fenafluramine was identified to be capable of targeting serotonin receptor subtypes, acting as an agonist for those subtypes and allowing serotonergic neurotransmission. However, this drug does not restore the sodium channels that are absent in GABAergic neurons. While this drug is effective at targeting the right subtypes, it does have some comorbidities. It happens to target the serotonin-2B receptor subtype, which leads to cardiac valve hypertrophy. Fenafluramine's off-target activity has made it unsuitable for use in treating Dravet's syndrome.

FISH, flourescent insitu hybridization is a molecular technique used to visaulize expression patterns of genes in a model organism. Knowing where a gene is expressed can give clues as to what role the gene plays in the organism. In fluorescent in situ hybridization, a fluroescent RNA probe is made and introduced into the organism, where it binds the appropriate gene sequence. As you can guess, the sequence of the RNA probe has to be complementary to that of the desired DNA sequence. Probe generation involves DNA extraction, polymerase chain reaction (PCR) to amplify the desired sequence, and then in-vitro transcription to turn DNA to RNA. The probe then has to be labeled. The actual in-situ hybridization process varies depending on the model organism used. In zebrafish, the whole process last about 4 days. The image of the expression pattern can be obtained by using confocal microscopy.

DNA extraction is an essential technique used in many scientific research endeavours. In my gene and genome analysis lab, BIO383H, we extracted DNA from a young Brachypodium distachyon leaf. DNA is found in the nucleus of cells. As such, we had to disrupt cells and tissues to obtain the DNA. First, we did that by mechanically grinding the leaves. Then we had to use detergent, found in the DNA extraction buffer, to dissolve the solubilize the plasma membrane and other membranes within the cell. Within the extraction buffer is also a metal chelating compund which locks away calcium and magnesium ions, allowing the denaturation of proteins. High temperature was also used to augment the efficiency of extraction buffer and hasten cellular disintegration. After breaking up cells and tissues, we had to prevent the degradation of DNA by DNAse enzymes. The metal chelating compound already helped us with this by locking away calcium and magnesium ions, which are essential co-factors for enzyme activity. Next, we had to remove undesirable products. We did this by rounds of centrifugation and then precipitating out the DNA  by using high levels of sodium and isopropanol. To truly purify the DNA, 70% ethanol was used to wash it. Then it was dissolved in T₁₀E₁ before being stored in a freezer.

Though GABAergic neurotransmission has been found to be most directly affected by the mutations underlying Dravet's syndrome, scientists have explored the potential of other neurotransmitters rescuing the phenotype, relieving mutant zebrafish larvae of epileptic seizures. One of the neurotransmitters that has shown some promise is serotonin (5-hydroxytryptamine). It's been revealed that increasing serotonin levels can reduce epileptic locomotor activity and brain activity. As such, the drug fenafluramine was identified to be capable of targeting serotonin recetor subtypes, acting as agonists for those subtypes and allowing serotonergic neurotransmission. However, this drug does not restore the sodium channels lost in GABAergic neurons. While this drug is effective at targeting the right subtypes, it does have some co-morbidities. It happens to target the serotonin-2B receptor subtype, which leads to cardiac hypertrophy. This has made the drug unfit for use as an effective treatment against Dravet's syndrome.

Dravet's syndrome is a rare, severe, genetic epileptic encephalopathy. Most cases of Dravet's syndrome are caused by a mutation in the SCN1A gene. This gene codes for a sodium channel, voltage gated type 1 alpha subunit. In recent studies, Dravet's syndrome has been attributed to mutations in GABAergic neurons; hence, GABAergic neurotransmission is compromised. GABA is a nuerotransmitter with vast inhibitory functions. As such, seizurelike locomotor behavior and epileptiform brain activity are signs of Dravet's syndrome. In humans, the presence of one mutant allele is sufficient enough for the expression of the mutant phenotype but in zebrafish, the model organism for studying neural networks and disorders, the mutant phenotype is only observed in larvae that have two copies of the mutant allele. 

Superficially, the specimen appears to be a light brownish color. The specimen has a segmented body, with about 10 segments in total. The specimen is bilaterally symmetrical along the anterior-posterior axis, meaning that if the specimen were to be folded along just this axis, two similar sides will be observed. The anterior end is the side that leads during forward motion; the posterior side is on the opposite end. Posteriorly, there are two dot-like structures called spiracles. They constitute the respiratory apparatus of the specimen. The ends of the specimen appear to be darker that the more medial segments. As such, the medial parts are more translucent. The specimen is responsive to visual stimulus (flashes of light), indicating the presence of visual-sensory structures. Relative to its surrounding, the specimen appears to move in a transverse wave-like manner while also employing some elements of longitudinal wave motion along its anterior-posterior axis. The internal organs of the specimen appear to be surrounded by exoskeleton. I presume that this gives the specimen a level of protection from predators, when in its natural habitat, and mechanical injury.
Along the abdominal region, there are hair-like structures. Perhaps these render sensory functions to the specimen.

The specimen has a segmented body, with about 10 segments in all. The specimen is bilaterally symmetrical along the anterior-posterior axis, meaning that if the specimen were to be folded along just this axis, two similar sides will be observed. The anterior end is the side on which the specimen undergoes forward motion; conversely, the posterior side is opposite. Posteriorly, there are two dot-like structures that are actually spiracles. They are the respiratory apparatus of the specimen. The ends of the specimen appear to be darker that the more medial segments. As such, the medial parts are more translucent. The specimen is responsive to visual stimulus (flashes of light), indicating the presence of visual-sensory apparatus. Superficially, the specimen appears to be a light brownish color. Relative to its surrounding, the specimen appears to move in a transverse wave-like manner while also employing some elements of longitudinal wave motion along its anterior-posterior axis. The internal organs of the specimen appear to be surrounded by exoskeleton.