Probe Development

With the completion of human genome program and the easy accessibility of human genome information, ISH

probes can now be easily selected and prepared. The essential information is provided in the following protocol. More detailed protocols can be obtained from standard molecular biology texts or technical manuals of commercial suppliers.

Bacterial artificial chromosomes (BACs) were selected from the genome browser (http://genome.ucsc.edu/ cgi-bin/hgGateway) and ordered from CHORI (http:// www.chori.org) or Research Genetics (http://www. resgen.com). Usually, two closely positioned BACs of 200 to 300 kb were used together as one probe. Thus, the final labeled probe covers about 400- to 600-kb genomic DNA. DNA of human insert from the BACs was prepared using standard protocol.[1] The probes were labeled by random priming with biotin or digoxigenin using a commercial labeling kit (Invitrogen, Co., Carlsbad, CA; Roche Applied Science, Indianapolis, IN). The labeled probes were purified using MicroSpin S-200 HR column (Amersham Biosciences, Piscataway, NJ), and the quantity and quality of labeled DNA were evaluated by gel electrophoresis or spectrophotometry (usually 5 mg from one labeling reaction). Cot-1 DNA (Invitrogen) was added to the labeled probe (10 times the quantity of labeled probe, e.g., 50 mg of Cot-1 for 5 mg of labeled probe). This anneals with, and blocks, the nonspecific repeating sequences, such as alu or LINE sequences.

The probes used for the two examples were prepared before the completion of the human genome program. The selection of probe was slightly different from the method described above. The probes for MYC translocation were YAC probes. The human DNA inserts in the YAC were isolated by pulsed-field gel electrophoresis. For the HER2 probe, the two BACs were selected by ''BLAST'' HER2 gene sequence in the National Center for Biotechnology Information (NCBI) database. The nonspecific repeating sequences were removed in the commercial version of the probe.[3] Thus, no blocking DNA, such as Cot-1 DNA, was added to the probe. All probes are available from Zymed Laboratories.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook


Post a comment