Speed: From sample to PCR in less than 15 minutes

Versatility: Rapid DNA extraction from a variety of sample types

Traditional methods of DNA extraction are time consuming and laborious or require specialized kits optimized for individual tissue types. KAPA Express Extract kits offer a convenient and efficient alternative for the routine extraction of DNA from a variety of tissue types, including buccal swabs, hair follicles, FFPE tissue, bone marrow, blood, blood spots on denim, and processed foods. KAPA Express Extract kits coupled with KAPA2G Robust HotStart ReadyMix (which contains a novel DNA polymerase tolerant of carryover inhibitors), significantly improves PCR success rates when amplifying from crude extracts.

DNA barcoding is rapidly gaining support as a quick, cost-effective and broadly acceptable tool for species identification. 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.

Fast DNA extraction and increased PCR success rates from FFPE tissue

DNA extracts were prepared from two different FFPE samples using KAPA Express Extract. Sample 1 was archived for >6 months and Sample 2 for >1 year. 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.

Routine extraction of DNA from a variety of blood sample types

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.

Frequently Asked Questions

1. PCR failure when using DNA extracted with the KAPA Express Extract Kit.

KAPA Express Extract Kits have been extensively tested for the extraction of DNA from a wide range of sample types. Depending on the sample type, various DNA damaging agents and/or inhibitors are likely to be present that can affect both the extraction process and the downstream PCR. Initially, optimize the PCR protocol using the following recommendations and then repeat the DNA extraction.

Optimization of PCR:

• KAPA2G Robust HotStart ReadyMix or KAPA2G Robust DNA Polymerase Kits are recommended for downstream PCR using KAPA Express Extract. The KAPA2G Robust DNA Polymerase has been engineered specifically for improved processivity and tolerance to common PCR inhibitors through a process of molecular evolution. The robustness of the KAPA2G Robust DNA Polymerase is particularly useful for increasing PCR success rates for crude extraction applications using KAPA Express Extract. A kit combining KAPA Express Extract for DNA extraction and KAPA2G Robust HotStart ReadyMix for amplification is available (kit codes KK7151 (100 reactions) and KK7152 (500 reactions)).
• Some crude DNA extracts may contain high concentrations of PCR inhibitors. If the use of KAPA2G Robust DNA Polymerase fails to yield acceptable results, it may be necessary to dilute the DNA extract prior to PCR. Repeat the experiment with a positive control sample (e.g. genomic DNA purified using a column-based kit) and a 10-fold dilution series of the DNA extract (prepared in TE or 10 mM Tris-HCl, pH 8.0 – 8.5). The DNA extracts may also be “spiked” with a known concentration of the positive control sample to determine whether the inhibitors may be diluted out, whilst retaining a high enough template concentration to support amplification of the target.
• Reduce the annealing temperature by 2 - 5 °C, and/or increase the annealing or extension time. Inhibitor carryover may affect primer annealing and/or the processivity of the DNA polymerase.
• Non-specific products, primer-dimers and/or smearing may occur as the result of RNA or degraded DNA originating from the presence of inhibitors in the sample. Modifications to the PCR cycling protocol (e.g. increasing annealing temperature, reducing annealing and/or extension time) and using a HotStart DNA polymerase may reduce non-specific amplification.
• DNA extracted from difficult sample types can be further purified using isopropanol or ethanol precipitation to remove inhibitors (for detailed protocols please contact support@kapabiosystems.com). DNA should be quantified by measuring the absorbance of the sample at 260 nm, diluted if necessary, and used directly in a PCR.

Optimization of the DNA extraction:

• The lysis step at 75 °C may be varied between 10 and 30 min, try a longer incubation time at 75 °C to maximize DNA output.
• For certain sample types decreasing the temperature of the lysis step from 75 °C to 60 °C improves the efficiency of the extraction. Lysis temperature can be optimized by performing a temperature gradient from 55 - 75 °C for 10 min followed by incubation at 95 °C for 5 min.
• Heat-inactivation of the KAPA Express Extract Enzyme for at least 5 min at 95 °C is essential, as carryover enzyme will degrade the DNA polymerase during PCR.
• The addition of certain sample types may decrease the pH below 7. DNA is rapidly degraded in low pH environments, especially at elevated temperatures during the lysis protocol. Test the pH of the extracted DNA after the lysis protocol is completed using pH paper. If the pH is <7.5 use the KAPA Express Extract Buffer at a final concentration of 1.5 – 2X. Alternatively a smaller amount of sample can be added to the initial extraction reaction.
• Typically DNA extracts do not have to be quantified prior to use in PCR, and quantification is not recommended, however it is possible to use a NanoDrop or equivalent to get an indication of the amount of contaminating factors. 2. What are the recommended applications for KAPA Express Extract Kits? KAPA Express Extract Kits are ideally suited for the extraction of DNA from crude samples for most PCR applications.

3. How long can extracted DNA be stored?

DNA extracted using the KAPA Express Extract Kit will degrade over time in 1X Express Extract Buffer and the presence of carryover inhibitors may increase the degradation process. Typically DNA extracted from ‘clean’ samples such as buccal swabs and hair follicles are stable at 4 °C or -20 °C for several weeks after extraction, but this can be reduced to days depending on the sample type. A 1:5 dilution of the DNA extract in TE Buffer is recommended for longer-term storage. This is done to ensure that the DNA is stored in a stable buffered environment. The dilution factor may be varied between 1:1 and 1:20, depending on the downstream application and yield of DNA. For downstream applications that are sensitive to EDTA, TE may be replaced with 10 mM Tris-HCl, pH 8.0 – 8.5. DNA extracts stored in this manner are typically stable for at least 6 months. For longer term storage (especially from samples with a high concentration of carryover inhibitors) it is recommended that the DNA is further purified using an isopropanol or ethanol precipitation to totally remove inhibitors (for detailed protocols contact support@kapabiosystems.com).

4. How many PCR reactions can be performed from a single DNA extraction reaction?

Depending on the sample type, a single extraction typically yields sufficient template DNA for 50 – 500 standard PCR reactions. Usually 1 - 2 μl of the extracted supernatant is used in a PCR reaction (50 - 100 PCRs). To determine how many PCR reactions can be achieved from a single extraction, prepare a serial dilution with TE or Tris pH 8.0 with a small volume of extracted sample, with each serial dilution perform a PCR to determine the optimal dilution factor.

5. What sample types are not recommended for use with KAPA Express Extract Kits?

Plant samples, degraded samples, samples stored for extensive periods of time and/or of unknown quality, etc... are not recommended for use with the KAPA Express Extract system.

6. Can the DNA extracted using KAPA Express Extract Kits be used directly in a qPCR?

DNA extracted using KAPA Express Extract Kits can be used directly in qPCR, however there are limitations. Primary limitations include: quenching of the fluorophore by carryover inhibitors, e.g. blood is well known to quench SYBR fluorescence; inhibition of the qPCR reaction; low concentrations of DNA from extraction protocol.

Recommendations for using KAPA Express Extract with downstream qPCR include:

• Perform a serial dilution of the extracted DNA with TE or 10 mM Tris pH 8.0. This will dilute any potential inhibitors, reducing PCR inhibition and quenching of the fluorophore. It is likely that some quenching will remain, and the DNA concentration is likely to be underestimated.
• Quenching of the fluorophore is dependent on the type used, try using a different fluorophore. KAPA SYBR® DNA Polymerase has been engineered to perform optimally in stringent real-time qPCR reaction conditions, and has reduced inhibition to SYBR® Green I dye and hence elevated SYBR® Green I concentrations can be used.
• The DNA extracts may also be “spiked” with a known concentration of the positive control sample to determine whether the inhibitors may be diluted out. Ideally a serial dilution is prepared from the KAPA Express Extract sample and the same DNA used at one concentration of the standard curve added to all the serial dilutions. This will give a clear determination of inhibitors (increasingly delayed Ct score across serial dilutions) or no DNA in the extracted sample (same Ct score as standard, even with undiluted sample).
• Attempt different lysis protocols (see above for recommendations) to maximize DNA output.

7. Is a xylene wash step still required when working with FFPE tissue?

A xylene wash step is not required when using the KAPA Express Extract Kit. After the centrifugation step the layer of wax is usually found on top of the buffer or on the side. The location of the wax layer is variable and dependent on the size of the sample. The wax is less dense than water and thus collects on the top. Where it collects is related to the angle of the rotor. The supernatant contains the DNA and is retrieved by puncturing through the wax layer on top. It is recommended to trim the excess wax around the sample using a sterile scalpel to leave only the tissue. Excess wax will not effect the extraction process, but will make it more difficult to recover the DNA. KAPA2G Robust DNA Polymerase is recommended for downstream PCR. The KAPA2G Robust DNA Polymerase has been engineered for improved processivity and tolerance to common PCR inhibitors present in FFPE tissue.

8. Is centrifugation a requirement prior to PCR?

Depending on the sample type it is possible to omit the centrifugation step after the extraction reaction. KAPA2G Robust DNA Polymerase is recommended for downstream PCR since sample debris and inhibitors will be present at a higher concentration and will almost always result in unreliable results if other DNA polymerases such as wild-type Taq are used. Pelleting the debris will result in a more reliable PCR. It is essential to pellet blood prior to use in downstream applications.

9. Can I use other manufacturers of Taq Polymerase with DNA extracted using KAPA Express Extract Kits?

Wild-type Taq Polymerase will work with DNA extracted from ‘clean’ sample types (e.g. buccal swabs, hair follicles) but will be unreliable when amplifying more challenging samples and templates. For the highest performance amplification, the use of KAPA2G Robust HotStart ReadyMix is recommended for all sample types.

10. Can the DNA extracted with KAPA Express Extract Kits be used with other products from Kapa Biosystems?

KAPA Taq DNA Polymerase, KAPA2G Fast DNA Polymerase, KAPA HiFi DNA Polymerase, and KAPA SYBR FAST qPCR Kits have all successfully been used with DNA extracted using KAPA Express Extract to amplify a range of amplicons. KAPA2G Robust DNA Polymerase is recommended for everyday use with DNA extracted with KAPA Express Extract due to improved performance in the presence of carryover inhibitors.

11. Can KAPA Express Extract Kits be used for RNA extraction?

The extraction process has been optimized for DNA. RNA will chemically degrade during the heating protocol and so is not recommended with KAPA Express Extract Kits.

12. How should the KAPA Express Extract kit be stored?

KAPA Express Extract Kits are shipped on dry ice or ice packs, depending on the country of destination. Upon receipt, store the entire kit at -20 °C in a constant-temperature freezer. When stored under these conditions and handled correctly, all kit components will retain full activity for at least six months from the date of receipt, or until the expiry date indicated on the kit.

KAPA Express Extract Buffer and enzyme may be stored at 4 °C for regular, short-term use (up to 1 week). Provided that it has been handled carefully and not contaminated, the kit components are not expected to be compromised if left (unintentionally) at room temperature for short periods of time (up to 24 h). Long-term storage at room temperature or 4 °C is not recommended. Please note that reagents stored above -20 °C are more prone to degradation when contaminated by the user; storage at such temperatures is therefore at the user’s own risk.