KAPA Express Extract Kits

Figure 2. DNA was extracted with KAPA Express Extract Kit from various samples obtained from mammals and fish. From each extract, 2 µL was used directly (without quantification) in a PCR containing KAPA2G Robust HotStart ReadyMix and primers for the ~650 bp cytochrome c oxidase I gene fragment. PCR products (10 µL) were analyzed in a 1% agarose gel. Sample origin and type is displayed above the gel. Reaction products were used directly in standard Sanger sequencing reactions using out-nested M13 primers (2 µL PCR product per 10 µL sequencing reaction). Sequence data was of a high quality and enabled the identification of each species. A section of the sequence trace from Seriola lalandi (Yellowtail amberjack) tissue is presented in the bottom panel.

Figure 3. DNA extracts were prepared from two different FFPE samples using KAPA Express Extract. Each extract was used directly (without quantification) in multiple PCRs containing KAPA2G Robust HotStart ReadyMix and primers for five different fragments (293 bp - 1 kb) of the EGFR gene (corresponding to exons 18 - 21 and 24). Results were compared to those obtained using the same reaction and cycling conditions but using 1 ng purified human genomic DNA as template. With the exception of the 1 kb exon 24 fragment from the older sample, yields and reaction efficiencies were comparable between the FFPE DNA extracts and purified genomic DNA. The PCR products generated from sample 1 were diluted 1:10 and used directly in standard Sanger sequencing reactions. Sequence data (bottom panel, Sample 1 exon 19 fragment) was of a high quality. The mixed sequence starting at the position marked with the arrow confirmed the presence of a 15-nt deletion associated with non-small cell carcinoma diagnosed in the patient from whom Sample 1 was collected.

Figure 4. Extraction and amplification of DNA from different blood sample types for detection of the HLA-B*27 allele. DNA was extracted from 12 human EDTA blood samples with KAPA Express Extract (top panel). 2 µL of each extract was added directly to a 25 µL PCR containing KAPA2G Robust HotStart ReadyMix and two primer sets. The internal control primer set targets a 429 bp fragment of the beta globin gene, whereas the second primer set targets a 141 bp fragment of the HLA-B*27 locus in a sequence-specific manner. Two of the 12 individuals tested positive for the HLA-B*27 allele associated with ankylosing spondylitis. Lanes C- and C+ represent HLA-B*27 negative and positive controls respectively (1 ng purified human genomic DNA as template). DNA was extracted from “Guthrie” cards, FTA cards, or FTA Elute cards (bottom panel) spotted with blood of individuals confirmed to be HLA-B*27 positive (+) or negative (-). DNA extraction and amplification conditions and controls (C- and C+) were the same as for the top panel.