Figure 1.
Standard and new PCR/restriction enzyme-based methods for α and β tryptase genotyping.
Genomic DNA extracts from HMC-1, Mac-6 and two SMC preparations were each tested for the presence of β and α-tryptase genes by three different methods, each using the same 5′ and 3′ PCR primer pair as described in Methods. When the 1017-28 bp amplimers were exposed to EcoRV, the α-tryptase amplimer yielded 678 and 339-40 bp fragments. After labeling with ethidium bromide, all such products were detected (upper panel). In contrast, only the high molecular weight amplimer and 339-40 bp fragment were visualized using either DY682- (middle panels) or digoxigenin- (lower panels) labeled 3′-primer. DNA sequence analyses of the restriction site are shown above the corresponding gel electrophoresis pattern.
Figure 2.
Sensitivity of standard ethidium bromide and new PCR amplimer detection methods for α/β-tryptase genes.
Purified gDNA from a SMC preparation having both α- and β- tryptase genes and from MAC-6 cells, in each case at doses ranging from 0.02 to 81.9 ng, were subjected to PCR and labeled using ethidium bromide (top panels), or to PCR with DY682- (middle panels) or digoxigenin- (bottom panels) labeled 3′-primer. In each case labeled 1017-28 bp amplimer bands are shown along with the net fluorescence in the corresponding plots, representing three independent experiments (data in S1 Table).
Figure 3.
α/β-Tryptase genotyping using EcoRV-digested amplimers.
EcoRV-digested amplimers were labeled with ethidium bromide after gel electrophoresis (top panels) or, during the final PCR cycle with DY682 (middle panels) or digoxigenin (bottom panels) labeled 3′-primer. gDNA from SMCs with known ββββ, βββα and ββαα genotypes, defined as 4∶0, 3∶1 and 2∶2 β:α ratios, were utilized. Experimentally calculated percentages of the β-tryptase gene were plotted against the known percentages of β-tryptase (n = 3, mean ± SD). Standard deviations were too small to visualize the error bars in the 50 and 75%β-Tryptase (theoretical) values for the middle plot. Data is stored in S2 Table.
Figure 4.
A comparison of potential enhancers on the PCR of α/β-tryptase gDNA using DY682-labeled (left panels) or digoxigenin-labeled (right panels) 3′-primer.
The effects of ethylene glycol (0.5 to 2.5 M), betaine (0.5 to 2.5 M), glycerol (0.5 to 2.5 M), DMSO (0.5 to 10%) or no additive was examined on PCRs performed with gDNA amounts of 0.3-0.5 ng and Taq DNA polymerase of 0.75 U per PCR with ethylene glycol or betaine, or 1.2–2.0 ng of gDNA and 1.25 U of Taq DNA polymerase per PCR with glycerol or DMSO. Representative images of the 1017-28 bp amplimers from three independent experiments are shown. Band intensity data is stored in S3 Table.
Figure 5.
Tryptase genotyping in healthy subjects.
α/β-Tryptase genotyping was performed with gDNA obtained from SMCs of 24 subjects. EcoRV digestions of PCR amplimers labeled during the final cycle with DY682-labeled 3′-primer were performed. Gel images are shown for 3 of the 24 samples analyzed, 2 with a β:α ratio of 3∶1 and 1 with a ratio of 2∶2. The 17 samples in which the α-tryptase gene was detected are shown in the plot, where those with a 2∶2 or 3∶1 genotype are grouped together. Data is stored in S4 Table.