Category Archives: Molecular Biology

DNA Sequencing Methods

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While DNA-sequencing methods are diverse and complex they can be grouped into three categories which share several common features: 1. DNA Fragmentation, 2. Fragment Amplification, 3. Sequencing via Fluorescent-Synthesis. These categories are:
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PCR Mutagenesis: Overlap Extension

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Polymerase Chain-Reaction (PCR) has become the backbone of most Methods in Molecular Biology and site-specific mutagenesis no exception. The key to PCR-based mutation of DNA is careful design of primers. In the simplest case, a point-mutation can be inserted into all PCR products by adding a point mutation to all primers. Unfortunately, for linear DNA, this method only works for mutagenesis at the ends of the template (where the primers bind).

Overlap extension, is a powerful 2-step, multi-PCR technique that can insert mutations at any position and of any size (including whole deletions or insertions). It accomplished this using chimeric primers to (1) cut out pieces of DNA and (2) reassemble them at overlap points:
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Model Organisms and DNA’s “Molecular Clock”

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“Model organisms” are the best-studied organisms in experimental biology. For a particular research questions a specific “model organism” is chosen for its balance of: (1) ease of use and (2) “generalizability” of results. For example

  • unicellular organisms(e.g. bacteria and yeast): are used to answer questions in basic biochemistry or molecular biology;
  • invertebrates (e.g. worms and flys): are used to answer questions in genetics or embryonic development
  • vertebrates (e.g. zebrafish to primates): are used in models of human disease (as they have requisite physiological and neurological complexity)

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The Maximal “Rate” of Evolution

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The timescales over which organisms “mutate” or “evolve” varies dramatically from months(viruses) to millions of years(animals). The figure above plots the average genome size in base-pairs (x-axis) versus the average mutation-rate(y-axis) for various organisms. In addition, a second y-axis (“minimum time to 1% mutation”) illustrate approximate timescales corresponding to each rate.

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Introduction to RT-PCR (gene/mRNA expression)

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Quantitiative Reverse-Transcriptase PCR (RT-PCR) is different from regular PCR in that it does not measure genes(i.e. DNA) per se but rather measures the EXPRESSION of those gene (i.e. mRNA). Given the fact gene expression (mRNA) can vary dramatically between cell-types, it is important to first isolate a single cell-type by:

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Introduction to PCR and Animal Genotyping

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In a follow up to our overview on DNA methods, we wanted to discuss PCR (polymerase chain reaction) which is one of the most sensitive and versatile techniques in molecular biology. PCR is a technique which selectively amplifies any targeted DNA from a complex mixture based on a set of framing primers. These primers are ~20 base oligonucleotides which we can (1) design based on a sequenced genome and (2) make/order based on solid-phase chemical synthesis. PCR has many applications (see partial list below) but is to test for a particular gene/mutation (i.e. “genotype”) in an animal (see figure above).

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Introduction to the “Family Tree” of DNA Methods

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Many methods in molecular biology are simply different combinations of a handful of techniques. These combinations can be represented as a “family tree” whose “trunk”/”backbone” is PCR (polymerase chain reaction) and whose “roots”/”foundation” is built upon: (1) chemical synthesis of short oligonucleotides, (2) fully sequenced genomes and (3) vectors derived from bacteria and viruses.

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Estimating Metabolite/Protein Concentrations from RNAseq Data

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Under steady-state conditions, it is possible to estimate the concentration of a metabolite from the amount of protein and the amount of protein from the amount of mRNA (see figure above). In general, the conversion factors used for these calculations are simply the ratio of the “first-order” formation and degredation rate constants for the protein/metabolite of interest. Recently, a paper published in Nature, characterized the distribution per gene of: (1) total mRNA and protein (2) rates of mRNA and Protein synthesis and (3) rates of mRNA and protein degradation (see figure below).

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