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BIOL 239 (290)

Set 21.docx

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BIOL 239
Christine Dupont

Set 21 3 DNA probesShort singlestranded stretches of DNA of known compositionFrom 25 to several thousand nucleotides in length32To visualize them the probes are labelled usually with P or fluorescent dyesUsed in the identification of clones that contain complementary DNA sequences4 screening libraries by hybridization Transfer colonies to a nylon membrane that fits right on top of the plateColonies will stick to the nylon membraneKill bacteria make it single stranded fix it to nylon so it doesnt wash off and then probe with DNA sequencesNylon membrane goes into NaOH solution It lyses the cells in situ stay stuck DNA goes from single to double stranded in NaOH Stays in the same position thoughDry it and the DNA will stay fixed to the filter in the same position it was on the original plate so now you can compare themPut nylon filter into glad sandwich bag add buffer as well as DNA probe red dots and incubate overnight and the probe will hybridize to complementary sequences it finds Next day wash it and do detection you cant see it directly so you have to expose it to light sensitive film autoradiographic film Light causes black dots to form Its on the same position as the original plate so thats where youre gene of interest is5 DNA probesseveral ways to make labeled probesCan be chemically synthesized and are termed oligonucleotides usually 25 to 100 nucleotides in length Fluorescent dyes can be added as part of the synthesis proceduremust know the DNA sequence of the probe6 DNA probesORCan label a previously cloned fragmentremoval from the vector by restriction endonuclease digestion followed by chemical labellingusually 100500 base pairs in lengthdont need to know the sequence of this probe7 requirements and limitations of hybridization probesThe region of complementarity between probe and target sequence must be long enough to allow sufficient hydrogen bonds to provide a cohesive force
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