Sensitivity of PCR versus hybridization of an internal probe to the first round product.
(A) Primary and semi-nested PCR demonstrating a sensitivity of one cell in 102 polyclonal cells after the primary round of amplification and one cell in 105 after the semi-nested second round. Primary PCR: serial 10-fold dilutions of 100 ng patient DNA derived from BM (approximately 104 cells) were mixed into 1μg polyclonal DNA (approximately 105 cells) extracted from normal PB mononuclear cells. Lane 1: positive control with 100 ng patient DNA, no polyclonal DNA. Lanes 2 through 8: log concentration of polyclonal versus patient DNA (eg, lane 2: polyclonal vs patient DNA is 100:1). Lane 9: negative control, 1 μg polyclonal DNA, no patient DNA. M: 100-bp markers. Semi-nested PCR: 1 μL 10−3 dilution of the first-round product serves as a template in the second round of amplification with the inner CDR-III clone-specific primer. Each lane corresponds to a dilution of the primary PCR product directly above it on the gel. Lane 10: no template from primary PCR. Anticipated product lengths were 101 bp and 75 bp for first- and second-round products, respectively. Lanes with an effective patient–polyclonal concentration of 10−6–10−8 are not expected to contain any malignant cells. The 350-bp product seen in lanes 6 to 9 reflects a nonspecific product amplified from polyclonal DNA with the clonotypic primers. (B) Demonstration of a sensitivity of 1 cell in 10−3 polyclonal cells after hybridization to a32P-labeled CDR-III inner primer. Hybridization of the probe to the semi-nested product serves as a positive control. The gel from Figure 3A was transferred by vacuum Southern blot and hybridized at 42°C to a 19-mer oligonucleotide in 6 × SSPE/1% sodium dodecyl sulfate (SDS). The filter was washed (6 × SSPE/1%SDS × 10 minutes × 3 at room temperature, followed by 1 × SSPE/1% SDS × 3 minutes at 42°C) and exposed overnight.
