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    DNA Sequence Variation, Epigenetics, and Gel Electrophoresis

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    Question 1: Describe DNA sequence variations that can be often observed among individuals of the same species. Discuss the possible phenotypic consequences of these DNA sequence variation, give examples, if possible. Discuss the technologies or methods that are often used in detect each of these variations.

    Question 2: Describe the field of epigenetics study. How do epigenetic modifications affect gene expression and phenotype?

    Question 3: Briefly describe the principles and applications of 2-dimensional gel electrophoresis, including 2d DIGE, for analysis of proteins.

    Question 4: How does real time PCR work to quantify the initial amount of the DNA template? How should the method be modified to quantify the initial amount of RNA template extracted from tissue or cells?

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    SOLUTION This solution is FREE courtesy of BrainMass!

    1) DNA variations within the same species can occur by a few different means, including translocations, polymorphisms, and amplifications/deletions. The most common of these (and probably the answer this question is looking for) is the polymorphism, also known as the single nucleotide polymorphism (SNP) or single nucleotide variation (SNV).

    There are several possible consequences of a SNP. Here are a few that I can recall:
    a) Nothing, sometimes a SNP is just there and acts in no capacity

    b) Mutation of the amino acid that is encoded by the gene. This assumes that the SNP is not in an intron (uncoded region of DNA), but it's one of the more dramatic possibilities. You can probably imagine pretty easily how bad a mutation can get, with the result being a damaged or dysregulated protein that needs to be taken care of by the cell. Ultimately, this could result in disease

    c) Frame shift - If your SNP accidentally changes an amino acid coding codon into a stop codon or something like that, then you can get a protein that is truncated. Half a protein is usually pretty useless!

    d) Transcriptional/Translational changes - Even if the SNP were to cause no change to the code of the protein, the body very tightly regulates transcription and translation. Changes to the coding sequence can change how fast things are made or degraded, though it is not possible (yet) to predict how this will affect rates because we do not have a clear understanding of HOW these processes are regulated

    The most commonly-used technology used to discover new SNPs is DNA sequencing, which is gaining in speed almost every day, it seems. For screening of known SNPs in individuals, we can use SNP arrays, but that assumes that common SNPs are present, because it only detects SNPs you are specifically looking for.

    2) Epigenetics is the regulation of DNA without regulating DNA sequence. Basically, we can make modifications to the histones (primarily by acetylation and deacetylation) that help wrap DNA into chromosomes or to the DNA itself (methylation and demethylation, primarily). These epigenetic changes can be accrued over an individual's lifetime due to environmental and genetic factors (e.g., smoking). The modifications made to histones and DNA change the accessibility by transcription factors and other molecules, though it is very context-specific whether any given modification will promote or inhibit transcription.

    This can have a dramatic impact on phenotype; epigenetics is one of the major ways in which the body controls what genes are expressed in certain locations. Have you ever wondered how a brain knows how to be a brain, while a liver with the same genome knows how to be a liver? Epigenetics plays a major role in that! Unfortunately, dysregulation of epigenetics can also lead to diseases like cancer, which is why this field is really hot in research right now.

    3) For this, I assume you already know what 1-d gel electrophoresis is, where we use SDS and a reducing agent to denature the protein and separate based on size. The second dimension adds an additional step before this. That first step is typically (though does not have to be) isoelectric focusing, where we first separate proteins based on the isoelectric point using a gradient pH gel. This essentially resolves them by charge, and then you can transfer that sample to a traditional 1-d electrophoresis setup and separate by size.

    The advantage is that it gives you MUCH higher capacity to resolve proteins. It's highly unlikely that two proteins will have the exact same molecular weight AND isoelectric point.

    Here is a URL with a really useful description and a picture of the data:

    4) Real-time PCR can use a few different methods to quantify protein. The simplest is the incorporation of DNA-binding dyes into the reaction. Basically, as you make more DNA strands using the PCR, the binding dye intercalates into the products. This allows the molecule to become fluorescent if you shine a UV light on it, so over time this fluorescence will increase. You can easily quantify this fluorescence with the right setup, allowing you to arrive at how much DNA you started with.

    For RNA, the principle is the same, except that we can't directly copy an RNA strand. First, we must reverse-transcribe it using a virally-derived reverse transcriptase. This generates DNA strands that we can then use for quantitative PCR.

    DISCLAIMER: I am a practicing biochemist/molecular biologist, so most of this information came from my head. I have found some URLs that could prove helpful to you, but I cannot guarantee they will contain exactly what I said here. Molecular biology is quite complex, and I am not an expert in any of these fields. For the purposes of your class, though, those explanations should be sufficient. Please let me know if I can clarify anything for you!

    On SNPs:

    On epigenetics:

    On Reverse Trascriptase-PCR:

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