We decided to use ASPCR in the development of an assay for noninvasive prenatal screening.
However, any delay in processing can lead to lysis of the intact cells with an increase in the proportion of maternal cell-free DNA in the samples, which can subsequently interfere with ASPCR.
After searching for a suitable method that would allow detection of fetal mutations in maternal blood samples, we decided to use ASPCR, a PCR-based technique often used for genotyping.
On the basis of our experience with ASPCR, it is possible to detect certain types of genetic alterations present at concentrations <5 copies in a PCR tube and simultaneously suppress up to ~1000 copies of the wild-type allele (3 ng of genomic DNA).
To test the sensitivity of DHPLC method for genotyping of the TPMT*2 and *3 alleles, the DHPLC results of all 98 individuals were compared with PCR-based genotyping data (PCR-RFLP and ASPCR) for the *2 and *3 mutations.
Usually, PCR-based methods (e.g., PCR-RFLP and ASPCR) have been used for the detection of TPMT mutations (16, 22).
 Nonstandard abbreviations: TPMT, thiopurine S-methyltransferase; RBC, red blood cell; RFLP, restriction fragment length polymorphism; ASPCR, allele-specific PCR; DHPLC, denaturing HPLC; and Hb, hemoglobin.
A total of 1369 samples were genotyped for the factor V (G1691A) Leiden mutation by both the Invader assay and ASPCR. A group of 290 unrelated Caucasian thrombosis patients whose blood had been referred to the Hemostasis Reference Laboratory of The Blood Center of Southeastern Wisconsin after a first or recurrent objectively confirmed deep vein thrombosis or pulmonary embolism was included in this evaluation.
ASPCR is a powerful technique for the discrimination of alleles arising from single or multiple base substitutions.
Complete concordance was observed between ASPCR and Invader factor V genotyping among the 245 wild-type (1691GG), 42 heterozygous (1691GA), and 3 homozygous (1691AA) thrombosis patients.
 Nonstandard abbreviations: PM, poor metabolizer; EM, extensive metabolizer; and ASPCR
, allele-specific PCR.
(1) Nonstandard abbreviations: DGGE, TGGE, denaturing, temperature gradient gel electrophoresis; ds, double stranded; ss, single stranded; SSCP, single-strand conformation polymorphism; HET, heteroduplex analysis; CCM, chemical cleavage method; EMC, enzyme mismatch cleavage; CFLP, cleavage fragment length polymorphism; PTT, protein truncation test; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RT, reverse transcription; ASO, allele-specific oligonucleotide; RFLP, restriction fragment length polymorphism; PODGE, profiling of oligonucleotide dissociation gel electrophoresis; ASA, allele-specific amplification; ARMS, amplification refractory mutation system; ASPCR
, allele-specific PCR; and OLA, oligonucleotide ligation assay.