Spring 09 lecture 15

Spring 09 lecture 15 - Analyzing Mixtures of DNA RNA or...

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Analyzing Mixtures of DNA, RNA or Protein
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DNA Gene - entire DNA sequence necessary for the synthesis of a RNA molecule or functional polypeptide. It includes coding and regulatory regions (up- and downstream) We want to find out (for example): If there is a particular gene (of interest) in a particular genome? How many copies of the gene are present in the genome? Location of the gene (which chromosome)? RNA/protein We want to find out (for example): Is the gene expressed or not? When is it expressed? Which tissues? Different size of transcripts (or proteins)? Is expression changed under certain conditions? Is change in expression of protein coded by gene of interest in correlation with particular disease? Information carried by a gene is converted into observable product (definition of gene expression)
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However, more then one question: 1. Is the gene of interest transcribed: yes no 2. If yes – is it transcribed more (then control): yes no 3. If yes – did we find more of the accumulated transcript (mRNA): yes no (as mRNA is getting degraded…) 4. If yes – is transcript viable and carried to the ribosomes for translation: yes no (as mRNA continues to get degraded…) 5. If yes – is the protein produced: yes no (as some conditions might not be right for its translation) 6. If yes – is the protein properly folded: yes no 7. If yes – is protein properly distributed and/or functional What do we measure/assess:
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Southern blotting Northern blotting Western blotting Methods depend on whether you are trying to detect DNA, RNA or protein Nucleic Acid Hybridization = means for detecting complementary DNA or RNA Southern blotting: DNA (capital S) detection northern blotting: RNA detection western blotting: protein detection Expression of our gene of interest is usually assessed by: - observing the abundance of RNA transcribed from gene of interest - observing the amount of protein translated from (this) mRNA
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Detection of Nucleic Acids and Proteins Four general questions / concepts / steps: 1. What do we need in order to detect the specific gene or specific product of gene expression (TARGET)? 2. What does it mean “labeled”? We need a really good labeled hybridization PROBE =stretch of nucleotides complementary to gene of interest (DNA; Southern) or mRNA for gene of interest (northern) Nucleotides, which will be incorporated into the probe, carry labels  they could be detected synthesized probe could be detected (visualized) probe will hybridize with target target will be detected In general: TARGET is unknown and PROBE is known to us
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3. How do we make (synthesize, construct…) a labeled probe? We need to have either a) whole or partial coding sequence for the gene of interest from the same organism, or b) whole or partial coding sequence for the gene of interest from the different organism (homologous gene), or c) sequence of the gene from the same gene family as our gene of interest This will be a starting point for making a very specific
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