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Alu-related approaches
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InterAlu-PCR and nick translation comparison
For large human probes (P1s, BACs, YACs) nick translation labeling can be replaced by interAlu-PCR labeling. For PCR, we used two primers, CL1 and CL2, previously published by Christoph Lengauer.
CL1
= 5' tcc caa agt gct ggg att aca 3'
CL2 = 5' ctg cac tcc agc ctg gg
3'
This PCR amplification is especially useful for YACs, as their DNA is usually isolated as a mixture of yeast and YAC DNA. Nick translation labels both DNAs, whereas interAlu-PCR specifically labels only the human DNA insert of the YAC molecule. As shown in Fig. 10e and 10f, both procedures work (red signals) with comparable results. When probes were labeled by interAlu-PCR it was very important to adjust the amount of competitor DNA to block out repetitive sequences. In Fig. 10e, a 4-5x larger amount of a green-labeled YAC probe was combined with another red-labeled probe. The competitor DNA competed out the red background but not the green background. As it is often the case with interAlu PCR, the background from the green-labeled DNA also provided a pseudobanding of the chromosomes, which can be used for chromosome identification. In Fig. 9f, repetitive signals were completely blocked out and signals were specific. This image also shows a common finding when YACs are used for FISH. Many clones, probably around 50% in the CEPH library, are recombinant, thus yielding specific and nonspecific signals (arrows) on different chromosome pairs. Although hybridizations with nick-translated and interAlu-PCR labeled YACs may yield similar FISH results, it is likely that interAlu-PCR does not really amplify the human sequence 100%, as the amplification depends on the density and orientations of the Alu repeats in the clone. Thus, small recombinant fragments may escape labeling and detection.
Methods of Alu banding
The biotin-labeled CL1 primer was synthesized and was hybridized onto normal human chromosomes (Fig. 10g). Posthybridization washing conditions were very gentle (in 2xSSC at 37° C). Results did not reveal a clear banding pattern, probably because the short primer yields only weak signals. By comparison, a Cy7-labeled 77mer Alu-primer or the labeled PCR products of interAlu-PCR reaction on any large probe could be successfully used for pseudobanding of the chromosomes (Fig. 10e). The 77mer primer and the PCR products probably hybridize on more defined, longer Alu sequences, and the banding pattern becomes visible. Alu banding can be used along during FISH with other probes, to help identify human chromosomes.
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