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Lecture 16

Lecture 16 - Analyzing Mixtures of DNA, RNA, or protein - BIOL308.pdf

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BIOL 308
Bruce Wolff

Lecture 16Analyzing Mixtures of DNA RNA or ProteinDNAGene entire DNA sequence necessary for the synthesis of a RNA molecule or functional polypeptide It includes coding and regulatory regionsupand downstreamWe want to find out for exampleIf there is a particular gene one of interest in a particular genomeHow many copies of the gene are present in the genomeLocation of the gene which chromosomeInformation carried by a gene is converted into observable product definition of gene expressionRNAProteinWe want to find out for exampleIs the gene expressed or notWhen is it expressedWhich tissues Different size of transcripts or proteinsIs expression changed under certain conditionsIs change in expression of protein coded by gene of interest in correlation with particular diseaseExpression of our gene of interestis usually assessed byObserving the abundance of RNAtranscribed from gene of interestObserving the amount of proteintranslated from this mRNAHowever there is more then one questionIs the gene of interest transcribed or not1If yesis it transcribed more then in control question of suitable control2If yesdid we find more of the accumulated transcript or not as mRNA is getting degraded3If yesis transcript viable and carried to the ribosomes for translation as mRNA continues to get 4degradedIf yesis the protein produced as some conditions might not be right for its translation5If yesis the protein properly folded6If yesis protein properly distributed andor functional 7Methodsdepend on whether we are trying to detect DNA RNA or proteinNucleic Acid Hybridization means for detecting complementary DNA or RNASouthern blotting DNA capital S detectionNorthern blotting RNA detectionProteinProtein InteractionWestern blotting protein detectionDetection of Nucleic Acids and ProteinsFour general questionsconceptsstepsWhat do we need in order to detect the specific gene or specific mRNA TARGET1We need a really good labeled hybridization PROBE stretch of nucleotides complementary to gene of interest DNA Southern or mRNA northern for gene of interestIn generale TARGET is unknown and PROBE is known to usWhat does it mean labeled2Nucleotides which will be incorporated into the probe carry labelsThey could be detectedSynthesized probe could be detected visualized NOTES FROM LECTURES Page 1
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