Increased maternal plasma fetal DNA concentrations in women who eventually develop preeclampsia.

was Ampli-Taq DNA polymer-ase. Another problem may be attributable to the production of uneven peak-height patterns, which are caused by differences in the efficiency of dideoxy termination at different bases and are affected by sequence context as well. Zakeri et al. (5) reported that a heterozygous C peak was much smaller in size than a heterozygous G peak. This is in agreement with our results. In addition to the production of uneven peaks, a high background, produced by impurities in the DNA template, could further complicate the results, especially for heterozygote detection. In our case, the background in the electropherograms was fairly low because all the 998-bp DNA fragments were purified by gel extraction. The quality of sequence data was actually very good for each sample, allowing us to read a sequence of ϳ500 nucleotides, which matched completely with the published NAT2 gene sequence (9). However, in a few samples, the signals produced by heterozygosity at the T 341 C and C 282 T sites were below the set default value (30%) for heterozygote detection. For example, our quality-control sample (sample 20) could be scored as a borderline heterozygote in the reverse se-quencing reaction (27% G and 100% A) but not with the forward primer. If the threshold value of heterozygote detection was set lower, e.g., 20%, then sample 10 could also be scored as a heterozygote, at least in one sequenc-ing direction. When a scoring problem arises for a sample after sequencing in both directions, it should be considered an undefined genotype until an independent method is used for its identification. We therefore used independent PCR-RFLP methods to check the genotypes of all 20 samples at three different polymorphic sites in the NAT2 gene, and discrepancies were found between the sequenc-ing and RFLP methods for only four test samples (Table 1, samples 8, 9, 10, and 11). In conclusion, there are some inherent problems in automated DNA sequencing, which may lead to inaccurate heterozygote identification in some samples. Until the associated problems are fully resolved, precautions should be taken in the use of automated sequencing for heterozygote detection. If possible, we recommend the use of two complementary PCR-RFLP methods in this type of analysis. height patterns in dichloro-rhodamine and energy transfer dye terminator sequencing. a single-nucleotide polymorphism by PCR-ELISA allele-specific oligonucleo-tide hybridization typing and by amplification refractory mutation system. Cloning and expression of cDNAs for polymorphic and monomorphic arylamine …