LAB5.NEW - Recombinant DNA Session 05 Nick Translation...

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Recombinant DNA Session 05: Nick Translation BACKGROUND Nick translation is a well-established technique for labeling DNA probes. A probe is a piece of nucleic acid labeled with a tracer ( radioactive or histochemical) that allows an experimenter to track the hybridization of the probe to an unknown DNA. The DNA used to make the probe is homologous to some or all the sequences contained in the digested plasmid DNA that you ran on your gel and transferred by Southern blotting. By labeling your probe and hybridizing it to the Southern blot, you can find out which DNA fragments on your gel your unknown probe is homologous to. The DNA to be labeled will be reacted with a small amount of DNase I and E. coli DNA polymerase I with DNA precursors (deoxynucleotide triphosphates). One of the deoxynucleotide triphosphates (a derivative of thymidine) is bound to the molecule digoxigenin, a large steroid molecule that can be recognized and bound very tightly by an antibody specifically raised against digoxigenin. Some of the existing DNA strands of the probe will be excised and repaired by the polymerase using the deoxynucleotide triphosphates as substrates. Nick translation is used with double-stranded DNA. DNA is mildly nicked with DNase I. The repair activity of E. coli DNA polymerase I is then used to label the DNA. The 5' to 3' exonuclease activity of pol I excises nucleotides, starting at the nick, and the 5' to 3' polymerase activity copies in labeled nucleotide substrates. The method is inexpensive and easy to do, but is sensitive to the exact length of the fragment to be nick-translated and requires a relatively large amount of DNA (good fraction of a microgram [µg]). The smaller the probe, the more DNase that has to be added, so that each fragment to be nick-translated gets a nick. A very big piece of DNA, like an intact lambda phage molecule, only needs one or a few nicks per 40 kb or so of DNA to be labeled. Reaction time and the amount of DNase added are thus critical . Too little reaction time and fragments won't get labeled. Too much reaction time and the DNase will degrade all the fragments after the labeling has peaked. [ Optional: The labeled DNA probe will be separated
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This note was uploaded on 05/03/2008 for the course BIO 2322 taught by Professor Spotswood during the Spring '08 term at The University of Texas at San Antonio- San Antonio.

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LAB5.NEW - Recombinant DNA Session 05 Nick Translation...

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