Submitted to: Colloquium on Domestic Animal Cytogenetics and Gene Mapping
Publication Type: Proceedings
Publication Acceptance Date: June 13, 1997
Publication Date: N/A
Technical Abstract:
Since the quantity of isolated DNA obtained from microdissected chromosomal subregions is femptogram amounts, it is necessary to use PCR to obtain sufficient amounts for further manipulation. The Primer-Linker approach described by Jinno et al. (1992) allows the amplification of smaller starting amounts of DNA than the Linker-Adaptor method, and it does not require pre-annealing of two oligonucleotides and phosporylation steps prior to the PCR. However, the original Primer-Linker method was designed for microscale enzymatic processing. We modified the original technique so that it could be carried out in an Eppendorf tube. Microdissected fragments were transferred directly to the microfuge tube containing a small volume of water which was subsequently removed on a speedvac. Protease and restriction digests were carried out, primer and linker were added separately at 10 uM and 1 uM concentration, respectively, followed by ligation and 45 rounds of high stringency PCR. PCR product was cloned into TA vector (Invitrogen). Insert PCR was used to evaluate size of cloned fragments and hybridization of dot blots with porcine Cot1 DNA revealed highly repetitive inserts. Finally, we modified the original technique to incorporate methanol:acetic acid fixation to allow G-banding whereas Jinno et al. described the use of a fixative that does not allow subsequent banding.
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