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.
However, a frequent cause of failure of ASPCR is uneven distribution of different alleles of the same gene in a sample, which leads to false-positive results produced by the abundant form of the gene.
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).
Thus, to make ASPCR reliable and useful for clinical noninvasive prenatal testing, we focused on improving the accuracy of allelic differentiation to the extent that interference by the wild-type allele would not be a limiting factor.
Considering the previously mentioned problems with the accuracy of ASPCR for allelic differentiation, we hypothesized that adding replication accessory proteins to our reactions would suppress potential false-positive results caused by high amounts of the wild-type allele.
1] 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.
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.
2%) and required repeat analysis using the same DNA isolation; all 16 samples ultimately generated robust signals and typed concordantly with ASPCR.
Nonstandard abbreviations: APC, activated protein C; FVL, factor V Leiden; and ASPCR, allele-specific PCR.
Total signal Genotype by ASPCR n G reaction A reaction Wild-type (1691GG) 1260 Mean 729 155 SD 183 35 Range 264-1307 16-344 Heterorygous (1691GA) 106 Mean 549 472 SD 143 170 Range 221-1000 126-1390 Homorygous (1691AA) 3 Mean 148 673 SD 11 51 Range 135-157 619-720 Net adjusted signal Genotype by ASPCR G reaction A reaction G:A ratio Wild-type (1691GG) 497 5 411 178 12.