Table 1.
Primer sequences and fragment sizes for pre-amplification and fast Temperature-Gradient COLD PCR.
Fig 1.
Fast Temperature-Gradient COLD PCR for rs7480526.
The input conventional PCR products resulted from spiked genomic DNA amplification consisting of 95% maternal alleles (blue) and 5% fetal alleles (red). First, the PCR products are subjected to a number of cycles of regular PCR to produce an initial pool of target amplicons. Next, the denaturation temperature is set to the first Tc, which is lower than the Tm of the maternal allele. Since the melting temperature of the fetal alleles is lower than that of the maternal alleles, fetal alleles get substantially denatured whereas the maternal alleles remain substantially double-stranded. Then we decrease the temperature for primer annealing and extension. Next, we move on to the second critical temperature (Tc2) and repeat the same procedure for ten cycles. In that way we span a range of three (SNP rs968857) or four (SNP rs7480526) critical temperatures and so we assure the preferential enrichment of the minor fetal allele and increase its abundance within the final product.
Table 2.
Critical temperatures of SNP rs7480526 and SNP rs968857.
Fig 2.
Electropherograms of spiked genomic DNA for SNPs rs7480526 and rs968857 using fast-COLD-PCR and fast-TG-COLD-PCR.
The top sequence above each electropherogram presents the reference sequence based on the GRCh37 genome assembly. Enrichment of the minor allele is observed using the fast TG COLD PCR whereas non enrichment is observed with fast COLD PCR for the same samples.
Fig 3.
Electropherograms of maternal plasma of families 182, 197 for rs7480526, rs968857 respectively, after fast-TG-COLD-PCR/conventional-PCR.
Three runs are indicated, 3 replicates per run, 9 reactions total. The top sequence above each electropherogram presents the reference sequence based on the GRCh37 genome assembly. Enrichment of the paternally inherited minor allele is observed in all replicates for SNP rs7480526 and in 8 out of 9 reactions for SNP rs968857 with fast TG COLD PCR as opposed to no enrichment with conventional PCR.
Fig 4.
Minor allelic frequency of the 17 maternal plasma samples analyzed for all the 141 replicates.
Blue columns: replicate samples with expected minor allele (Mother: GG/ Fetus: GA or GT). A minor allelic frequency distribution between 21–100% was observed in 101 out of the 105 replicates. A minor allelic frequency between 0–10% was observed in 2 out of 105 replicates and 11–20% in 2 out of 105 replicates. Red columns: replicate samples with no expected minor allele (Mother: GG/ Fetus: GG). A minor allelic frequency below 10% was observed in 33 out of the 36 replicates. A minor allelic frequency between 11–20% was observed in 2 out of 36 replicates and 61–70% minor allelic frequency in 1 out of 36 replicates.
Table 3.
Maternal plasma analysis results of SNP rs7480526 and SNP rs968857.
Three runs and three replicate reactions per run for eight and nine maternal plasma samples respectively.