John M. S. Bartlett.pdf - Bio-Nica.info

252 Ringquist et al.
2. The present method is based closely on the protocol described by Trenkle et al. (12,14),
in which RAP-PCR was used to generate probes for differential screening of cDNA
arrays on nylon membranes. Each array contained 18,432 cDNA clones from the IMAGE
consortium and hybridization detected approx 1000 cDNA clones using each RAP-PCR
probe. Different RAP-PCR fingerprints gave hybridization patterns having very little
overlap (less than 3%) with each other or with hybridization patterns from total cDNA
probes. Thus, repeated application of RAP-PCR probes allows a greater fraction of the
message population to be screened on this type of array than can be achieved with a
radiolabeled total cDNA probe.
3. In general, there are no constraints on the primers except that they contain at least a few
C or G residues, that the 3′-ends are not complementary with themselves or the other
primer in the reaction, to avoid primer dimer, and that primer sets are chosen that are
different in sequence so that the same parts of mRNA are not amplified in different
fingerprints. PCR with a combination of arbitrary and oligo(dT) primers, as has been used
in differential display (17–20), can also be used to generate an effective probe for cDNA
arrays. The use of different oligo(dT) anchor primers with the same arbitrary primer results
in considerable overlap among the genes sampled by each probe. This can be avoided by
using different arbitrary primers with each oligo(dT) anchor primer (13).
4. Total RNA as well as poly(A) purified mRNA can also be labeled during reverse transcription
for microrray analysis. Methods for preparing fluorescent cDNA from RNA have been
presented by Eisen and Brown (16).
References
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Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 20, 4965– 4970.
2. Vogt, T. M., Welsh, J., Stolz, W., Kullmann, F., Jung, B., Landthaler, M., et al. (1997)
RNA fingerprinting displays UVB-specific disruption of transcriptional control in human
melanocytes. Cancer Res. 57, 3554–3561.
3. Vogt, T., Stolz, W., Welsh, J., Jung, B., Kerbel, R. S., Kobayashi, H., et al. Overexpression of
Lerk-5/Eplg5 messenger RNA: A novel marker for increased tumorigenicity and metastatic
potential in human malignant melanomas. Clin. Cancer Res. 4, 791–797.
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by transforming growth factor beta 1. Proc. Natl. Acad. Sci. USA 90, 10,710–10,714.
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8. Welsh, J., Petersen, C., and McClelland, M. (1991) Polymorphisms generated by arbitrarily
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DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic
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