308 Lecture 16

308 Lecture 16 - Analyzing Mixtures of DNA, RNA or Protein...

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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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Southern blotting northern blotting western blotting Methods depend on whether we 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 Alberts: Mol. Biol of the Cell, 5 th edition
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However, there is more then one question: 1. Is the gene of interest transcribed or not 2. If yes – is it transcribed more then in control ( question of suitable control ) 3. If yes – did we find more of the accumulated transcript or not ( as mRNA is getting degraded…) 4. If yes – is transcript viable and carried to the ribosomes for translation (as mRNA continues to get degraded…) 5. If yes – is the protein produced ( as some conditions might not be right for its translation ) 6. If yes – is the protein properly folded 7. If yes – is protein properly distributed and/or functional 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 mRNA (TARGET)? Possible problem: target is sometimes present in very small quantities 2. What does “labeled” mean? We need a really good labeled hybridization PROBE 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 a suitable
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308 Lecture 16 - Analyzing Mixtures of DNA, RNA or Protein...

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