Hybridization Probe Selection

The hybridization probe must hybridize only with the unique DNA region of interest. The size of the hybridization probe selected should be between 300 and about 800 bp. Probes shorter than about 300 bp may require several day-long film exposures to achieve a suitable signal. The larger the probe is, the more likely it will pick up repetitive DNA elements, which will give nonspecific hybridization. A BLAST (blastn, Use MegaBLAST) alignment with the mouse genome can be used to determine whether the chosen probe sequence is sufficiently unique, before oligonucleotides for PCR are ordered or before sub-cloning is performed. Ideally, the sequence chosen should give a single BLAST hit (red line) with the following parameters: Expect = 0.01, Filter = none, Descriptions = 500, Alignments = 500. Sometimes the presence of a few low-score alignments can be tolerated. However, when thousands of hits occur, the sequence should not be used. Hence, this computational method at least eliminates potential probe sequences containing repetitive DNA elements that are extremely abundant in genomic DNA.

It is essential to experimentally assess the probe specificity under the exact hybridization conditions used to detect the nucleosome ladders. Nonspecific hybridization will still detect ladders, but they will represent the superimposed ladders arising from hundreds or thousands of loci in addition to the locus of interest. Most likely, the ladders detected with nonspecific hybridization will resemble those detected by ethidium bromide staining of the gel. Unfortunately, nearly half of the probes that we make do not hybridize with high specificity, despite use of the BLAST test. To assess the probe specificity, we include a lane on the gel (and the blot) containing a restriction enzyme digest of purified mouse genomic DNA. The probe should ideally detect only the predicted restriction fragment(s), without detecting a large number of other fragment sizes or a background signal consisting of a continuum of fragments. Figure 2 shows an example of a specific hybridization (Fig. 2A) and a nonspecific hybridization (Fig. 2B). For Fig. 2A the probe was 740 bp, and the BLAST test gave a unique alignment. For Fig. 2B the probe was 608 bp, and the BLAST test detected 9039 hits. The restriction digest of purified mouse genomic DNA (lanes D) shows detection of the unique predicted 740-bp fragment for Fig. 2A, but a high background continuum of fragments over the predicted 2.2-kb fragment for Fig. 2B. Additionally, the intensities of the ladder bands for Fig. 2B are unusually high for an overnight film exposure (compare with Fig. 2A). In this case, the BLAST predictions were confirmed experimentally.

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