Reliable DNA amplification is critical to the success of many molecular biology methods, from simple cloning and genetic screening to gene editing and next-generation sequencing (NGS). The DNA polymerase used can have a profound effect on the speed and reliability of the project. Among the key polymerase parameters that may have an impact are how well the enzyme can handle long reads, how fast and how accurately it copies the template, whether it has difficulties with repetitive, high GC- or high-AT content DNA, and whether it is particularly susceptible to chemical inhibition.
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In this podcast, Elizabeth Carpenter, Senior Support Scientist at Roche Sequencing, explains the benefits of KAPA HiFi DNA Polymerase and how it can help capture sequencing data from rare transcripts and low-copy variants.
The typical DNA polymerase offers either accuracy or speed, range, and the ability to tolerate inhibitors. But these attributes are not generally all found in the same enzyme, necessitating that choices must be made.
This may be fine for some routine work on run-of-the-mill samples. Even then, though, it’s best not to have to make such trade-offs. And especially when performing crucial procedures that require high-fidelity amplifications of long or difficult targets, or when dealing with precious samples, using the best reagents can end up reducing errors and thus avoiding headaches and saving time and resources.
Traditional applications as well as newer applications can benefit from a DNA polymerase that exhibits both high fidelity and high processivity. For example, in DNA cloning workflows, the ability to accurately amplify longer templates will help to reduce the number of clones needing to be screened for correct, error-free PCR-generated insertions, which is especially important for expression vectors.
Meanwhile CRISPR-based genome editing is only effective when the sequences of the guide RNAs (and repair templates) are correct, necessitating a polymerase able to reliably carry out the task.
Similarly, with NGS library preparation, a high-fidelity, high-processivity polymerase allows researchers to accurately capture the entire genomic complexity of the original samples for more uniform sequencing coverage.
KAPA HiFi DNA Polymerase has been engineered to combine both high fidelity and high processivity, enabling scientists to amplify long fragments with minimal artifacts, increasing confidence in cloning and other PCR-based applications, and of course NGS library amplification.
Figure 1 shows that the KAPA HiFi DNA Polymerase outperforms the competition when it comes to the ability to accurately reproduce its template. Due to its robust proofreading abilities, it is able to deliver lower error rates—averaging 1 per 3.6 x 106 nucleotides—than even other high-fidelity polymerases. This is of special importance in amplification of NGS library molecules, ensuring that identified variants are true sequence changes rather than artifacts of imprecise PCR.
Yet at the same time, KAPA HiFi DNA Polymerase excels in processivity, allowing amplification of targets up to 11 kb in length at high speed, and benefitting many different workflows. Accurate amplification of longer templates reduces the number of PCR reactions in DNA cloning workflows, ultimately reducing the number of steps and time-to-results, as well as conserving resources.
Templates rich in GC or AT base pairs pose a challenge to many polymerases, introducing amplification biases and even in some cases causing the enzyme to stall.
KAPA HiFi DNA Polymerase efficiently amplifies both GC- and AT-rich regions, making it ideal for preparing next-generation sequencing libraries capturing the entire genomic complexity, for more uniform sequencing coverage, and lower sequencing costs.
Figure 2 shows that NGS libraries amplified with KAPA HiFi DNA Polymerase display uniform coverage virtually indistinguishable from that of libraries obtained with a PCR-free workflow. In comparison, libraries amplified by other high-fidelity polymerases displayed a marked bias by underrepresenting coverage of GC- and AT-rich sequences.
Sensitive, accurate, full-length transcript coverage is needed to create high complexity libraries that yield excellent mapping and alignment metrics over a broad range of input. As such, KAPA HiFi DNA Polymerase is a common choice for many NGS library preparation workflows.
For example, the developers of the streamlined Smart-seq2 workflow—in which single-cell RNA is converted to cDNA and then pre-amplified—used KAPA HiFi HotStart polymerase to preserve the complexity of single-cell RNA in the input samples.1,2
Similarly, KAPA HiFi DNA Polymerase plays a prominent role in CIRCLE-seq, a highly sensitive NGS-based method to screen for off-target CRISPR/Cas9 editing, allowing scientists to ensure that detected mutations are not actually amplification errors.3
KAPA HiFi DNA Polymerase (or KAPA HiFi HotStart polymerase) is a key component of all KAPA Library Preparation Kits (Figure 3). The KAPA HiFi portfolio also includes KAPA HiFi Uracil+ for epigenomics research, engineered to amplify bisulfite-treated DNA templates with greater efficiency and fidelity.
No matter the amplification application, there is a KAPA HiFi-based product that fits the bill. To learn more, visit the Roche Sequencing USA site at Roche Sequencing Site-KAPA HiFi Kits
For Research Use Only. Not for use in diagnostic procedures.
1. Picelli, S., Faridani, O., Björklund, Å. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171–181 (2014). https://doi.org/10.1038/nprot.2014.006
2. Picelli, S., Björklund, Å., Faridani, O. et al. Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10, 1096–1098 (2013). https://doi.org/10.1038/nmeth.2639
3. Tsai, S., Nguyen, N., Malagon-Lopez, J. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nat Methods 14, 607–614 (2017). https://doi.org/10.1038/nmeth.4278