Kapa Biosystems Taq DNA Polymerase and PCR Kits

Figure 1.A 500 bp fragment of the CCR5 gene was amplified using 100 ng, 10 ng, 1 ng, or 100 pg of human genomic DNA as template. KAPA Taq HotStart exhibits improved sensitivity, specificity, and yield when compared with competitive HotStart products. All reactions were performed using the manufacturer recommended protocols.

Figure 2.A 270 bp amplicon was amplified from mycoplasma DNA with KAPA Taq or KAPA Taq HotStart. Sensitivity was tested using a 10X template dilution series starting with 1 ng of DNA.

Figure 3 .*Multiplex PCR (8-plex) performed with the KAPA2G Fast Multiplex PCR Kit, Competitor Q, and Competitor I. Reactions (25 μL) contained 1X PCR Master Mix (KAPA and Competitor Q) or 1X PCR Buffer, 3 mM MgCl2, 0.2 mM of each dNTP and 1 U of hot start Taq DNA Polymerase (home-brew multiplex reagents, with Competitor I). Human genomic DNA was used as template (250 – 2 ng per reaction), and primers were supplied at 0.2 μM each. Cycling was performed according to manufacturers’ recommendations (30 cycles).

Figure 4.*Multiplex PCR (12-plex) performed with the KAPA2G Fast Multiplex PCR Kit, Competitor Q, and Competitor I. Achieving uniform representation of all amplicons in a complex multiplex assay is a challenge due to amplification bias— a result of differences in amplicon length, secondary structure, and priming efficiency. Reactions (25 μL) contained 1X PCR Master Mix (KAPA and Competitor Q) or 1X PCR Buffer, 3 mM MgCl2, 0.2 mM of each dNTP and 1 U of hot-start Taq DNA Polymerase (home brew multiplex reagents, with Competitor I). Human genomic DNA was used as template (250 – 2 ng per reaction), and primers were supplied at 0.2 μM each. Cycling was performed according to manufacturers’ recommendations (30 cycles).

Figure 5.Fast Multiplex PCR with KAPA2G Fast Multiplex PCR Kits. Multiplex PCR with wild-type Taq typically requires long annealing and extension times to allow primer annealing and extension of all primers in the multiplex. Total PCR cycling times required for 4-plex, 8-plex, and 12-plex multiplex PCRs (30 cycles, set up according to the manufacturers’ recommendations) with KAPA2G Fast Multiplex PCR Kits and Competitor Q Multiplex PCR Kit (which contains wildtype Taq DNA polymerase) are shown. Time savings of 40 – 60% are possible with the KAPA2G Fast Multiplex PCR Kit.

Figure 6.GC-rich Multiplex PCR (6-plex) performed with the KAPA2G Fast Multiplex PCR Kit, Competitor Q and Competitor I. Successful Multiplex PCR with wild-type Taq is limited to easy, simple targets that can be amplified with equal efficiency. The improved processivity of the engineered KAPA2G Fast DNA Polymerase allows uniform multiplex PCR of a broad range of difficult targets. Reactions (25 μL) contained 1X PCR Master Mix (KAPA and Competitor Q) or 1X PCR Buffer, 3 mM MgCl2, 0.2 mM of each dNTP and 1 U of hot start Taq DNA Polymerase (home brew multiplex reagents, with Competitor I). Human genomic DNA was used as template (250 – 2 ng per reaction), and primers were supplied at 0.2 μM each. DMSO (5%) and KAPA Enhancer 1 (1X) was added to all reactions. Cycling was performed according to manufacturers’ recommendations (30 cycles). Amplicons range in size from 241 – 642 bp, and in GC content from 72.7 – 83.8%.

Figure 8.Amplification of a 1.5 kb fragment from 1 pg plasmid DNA in the presence of four common PCR inhibitors using the KAPA2G Robust HotStart PCR Kit and wild-type hot start Taq polymerase. All reactions contained 0.5 units of enzyme per 25 μL reaction. KAPA2G Robust HotStart Buffer B was used throughout, with the addition of KAPAEnhancer 1 for reactions containing SDS. Cycling was performed with an Eppendorf Mastercycler epgradient S, using a standard 3-step cycling profile (35 cycles) with an annealing temperature of 64ºC and 1.5 min extension time per cycle for all enzymes.

Figure 9.Amplification of a cloned 2.7 kb insert from four commonly used E. coli strains (DH5a, DH10B, JM109, or BL21) using KAPA2G Robust HotStart (top) or wildtype Taq (bottom). Colonies (grown on LB-agar + Amp plates) were either resuspended directly in individual PCR reactions (left) or first resuspended in PCR grade water and then added to PCR reaction mixes (middle). For overnight cultures (prepared in LB + Amp), 1 μ L was added directly to the PCR mix (right).

Figure 10.Amplification of a 2.5 kb (left) or 1.6 kb (right) fragment from the GSH1 gene from three commonly used S. cerevisiae strains (BY4742, FY23, and W303) using KAPA2G Robust HotStart (top) or wildtype Taq (bottom). Colonies (from YPD-agar plates) or YPD overnight cultures were first lysed in 50 μ L volumes with NaOH or Zymolase (as indicated).

Figure 11.Amplification of template dilution of human genomic DNA starting with 50 ng, 10 ng, 2 ng, 400 pg per 25 μL. Amplicons were 737 bp, 1.3 kb, 3.3 kb, and 4.5 kb in lengths. 35 cycles, 72ºC extension temperature.

Figure 12.Amplification of template dilution of human genomic DNA starting with 50 ng, 10 ng, 2 ng, 400 pg per 25 μL. Amplicons were 7.5 kb, 10.5 kb, 13 kb, and 15 kb in lengths. 35 cycles, 68ºC extension temperature.

Figure 13.*Amplification of 4.5 kb fragment from 50 ng, 10 ng, 2 ng, and 400 pg of human genomic DNA per 25 μL reaction. Lanes: (M) Marker, (1 – 4) KAPA Long Range HotStart, (5 – 8) Competitor T, (13 – 16) Competitor R.

Figures 14 and 15.Compared to the Quanta Mouse Genotyping Kit, KAPA Mouse Genotyping Kits exhibit better sensitivity, yield, and specificity in multiplex PCR. Reactions were performed with annealing at either 50°C or 60°C to demonstrate the effect of using sub-optimal annealing temperatures in genotyping assays.

Figures 18 and 19.DNA was extracted with KAPA Express Extract 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 commonly used in species identification (Ivanova, et al., 2007). 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 20.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 21.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.

Figure 22.Direct PCR using the KAPA3G Plant PCR Kit outperforms CTAB extraction and standard PCR using wild-type Taq, in significantly shorter turnaround times.

Figure 23.argets of different lengths (297 bp and 4100 bp from tobacco, 640 bp from tomato, 1221 bp from grapevine, and 1448 bp and 2249 bp from potato) were amplified from purified DNA (+) or leaf discs (L) using the KAPA3G Plant PCR Kit. For each species, genomic DNA was purified using a commercial DNA purification kit. Crude material was sampled using a 0.5 mm diameter Harris Uni-Core™ sampling tool. Purified DNA (1 – 10 ng per reaction, depending on species) and crude samples were used as templates in 50 μL PCRs, with 40 cycles of amplification. Reaction products were analyzed on a 1% agarose gel. KAPA Express DNA Ladder (100, 200, 400, 800, 1600, 4000, 8000 bp) was used as a MW marker.

Figure 24.Plant genomic DNA was purified from all species using a commercial DNA purification kit. A Harris Uni-Core™ sampling tool (0.5 mm diameter) was used to sample leaves (all species) or seeds (all species except tobacco and Arabidopsis; for these, one crushed seed was used per reaction). PCRs (50 μL) contained the crude sample or 1 – 10 ng purified DNA (depending on the species), and 40 cycles were performed in all cases. Targets ranged between 500 and 900 bp, and reaction products were analyzed in a 1% agarose gel. KAPA Express DNA Ladder (100, 200, 400, 800, 1600, 4000, 8000 bp) was used as a MW marker.

Figure 25.Crude sample PCR with the Tab c/d primer pair was performed on leaves and/or seeds from 8 plants, with 1 μL of crude extract prepared without heat treatment (left) or 1 μL of crude extract prepared with heat treatment (right). Reactions were set up as described in the KAPA3G Plant PCR Kit TDS, and 45 cycles of PCR performed with annealing at 55°C, and 20 sec extension at 72°C per cycle. KAPA Express DNA Ladder (100, 200, 400, 800, 1600, 4000, 8000 bp) was used as a MW marker. 1: Eucalyptus; 2: Grapevine; 3: Wheat leaf; 4: Wheat seed; 5: Canola leaf; 6: Canola seed; 7: Rice seed; 8: Barley seed; 9: Corn kernel; 10: Cotton seed; 11: Cotton leaf.