Genomic DNA Probes,Molecular Probes,Genomic DNA,Chimeric Vector

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	Genomic DNA Probes - Genomic DNA probes. Simple, reproducible methods have been devised for the preparation of molecular probes. A random or specific DNA sequence (probe) can be isolated from the genomic DNA (DNA extracted from cell nuclei), using following steps: (i) extract DNA from plant or animal tissue (ii) digest extracted DNA with a restriction enzyme (e.g. EcoRI or Hind III), which cuts DNA on specific sites or positions where a specific sequence recognized by the enzyme is found (iii) run the digested DNA on an agarose or polyacrylamide gel for. electrophoresis, to separate fragments of different sizes
(iv) isolate DNA of specific fragment size from a particular band identified through southern blots by hybridization with specific labelled mRNA or cDNA molecules, if available. If random probes are needed, no hybridization of Southern blots is required; Southern blots are DNA bands transferred from gel to nitro cellulose membrane with the help of a buffer rising through capillary action (v) clone this DNA in a vector (vi) allow chimeric vector to infect bacteria for multiplication, where it can make billions of copies.The steps (ii) to (vi) above are also used for preparing cDNA probes. From the transformed bacteria, the chimeric vector can be obtained and used in one of the following ways:
(i) It may be used directly as probe (the presence of vector DNA will not interfere in probe assays, so that it is not really necessary to remove it). (ii) The cloned segment maybe separated (or retrieved), by using the same enzyme which was used for cloning. In the latter case, cleaved chimeric vector DNA will be again electrophoresed for separating the inserted segment on the gel. This inserted segment thus retrieved can now be used as a probe. (iii) The chimeric DNA may be used for PCR, using flanking sequences as primers; the PCR product can be separated and used as a probe