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. 2002 Apr 16;99(8):5261–5266. doi: 10.1073/pnas.082089499

Figure 4.

Figure 4

Southern blot analysis of the human parathyroid hormone (PTH) and thyroglobulin genes and RFLP marker D13S2 for MDA-generated DNA target. (A) MDA reactions included a heat denaturation step and amplification was carried out as described. EcoRI DNA digests were probed by using a radioactively labeled genomic fragment of the PTH gene (p20.36) that hybridized to an ≈1.9-kb DNA fragment. The EcoRI-cleaved DNA preparations were genomic DNA, DNA amplified by MDA from varying amounts of input genomic DNA, as indicated, or an MDA reaction that lacked input genomic DNA template. The position of the 1.9-kb genomic DNA fragment is indicated (lane 1). Genomic DNA is shown: (lane 2) ×100, (lane 3) ×1,000, (lane 4) ×10,000, (lane 5) ×100,000, (lane 6) ×1,000,000, and (lane 7) 0 template. (B) MDA reactions included or omitted a heat denaturation step, as indicated, of genomic target DNA heterozygous for two thyroglobulin alleles, and amplification was carried out as described. TaqI DNA digests were probed by using a radioactively labeled genomic fragment of the thyroglobulin gene (pCHT.16/8) that hybridized to invariant 1-kb and 3-kb DNA fragments and a polymorphic 5.8-kb (allele A) or 5.2-kb (allele B) DNA fragment. The TaqI-cleaved DNA preparations were (lane 1) genomic DNA, (lane 2) DNA amplified by MDA reaction (×10,000) with a 95°C preheating step, and (lane 3) an MDA reaction (×10,000) without the preheating step. (C) Reactions lacked a heat denaturation step, but otherwise amplification was carried out as described. PstI DNA digests were probed by using a radioactively labeled genomic fragment of the RFLP marker D13S12 locus (p9D11) that hybridized to an invariant 3.8-kb DNA fragment and a polymorphic 2.1-kb (allele A) or 1.1-kb (allele B) DNA fragment. The PstI-cleaved DNA preparations were genomic DNA and five different patient DNAs amplified by MDA (×10,000 amplification): (lane 1) patient 5 (AB), (lane 2) patient 4 (BB), (lane 3) patient 3 (BB), (lane 4) patient 2 (AB), (lane 5) patient 1 (BB), and (lane 6) genomic DNA (AB). AB and BB represent the diploid genotypes of the D13512 locus.