Whole genome amplification (WGA) with less bias making this kit ideally suited for next generation sequencing applications.
The TruePrime™ WGA Kit uses a revolutionary multiple displacement amplification method based on the combination of the recently discovered DNA primase “TthPrimPol” and the extremely processive and high-fidelity Phi29 DNA polymerase to uniformly amplify genomic DNA from purified starting material. The extraordinary strand displacement capacity of the Phi29 DNA polymerase allows TthPrimPol to generate new primers on the displaced strands that are extended by Phi29 DNA pol, resulting in exponential isothermal DNA amplification. The typical DNA yield is >5 µg from a single reaction using 1 ng of starting DNA.
1 pg of human genomic DNA (~ 1/6 of the content of one human/mammalian cell) has been amplified using either TruePrime™ (TthPrimPol-based MDA) or random primed MDA reactions. Random primed reactions contain 20% of sequences that cannot be mapped to any organism in sequence databases.
Due to the high sensitivity of single cell whole genome amplification techniques there is a considerable danger of contaminating reactions with non-target derived DNA. We therefore recommend to take extreme care when setting up single cell amplification reactions as laid out in the handbook. However, TruePrime™ users have a unique advantage here: TruePrime™ does not accept non-denatured DNA strands as template as readily as random primed MDA reactions. Therefore, contaminations coming in from the environment etc. after the denaturation step will not have a strong influence on the composition of your reaction output. We have shown this behaviour in an experiment where we simulate an external DNA contamination by deliberately adding in yeast DNA to a denatured human DNA template. Yeast DNA is in a thousand-fold excess over the target DNA (1 pg human genomic DNA):
Whereas with TruePrime™ the vast majority of the sequences obtained are target-derived, random primed MDA shows a majority of contaminant-derived sequences.
We have amplified and sequenced the yeast genome using TruePrime™ and Illumina technology. Using 2.5 million read pairs we cover 99.4% of the total yeast genome. Shown is the coverage with narrow width of the distribution curve.
A coverage graph of yeast chromosome 7 exemplifies the more even coverage obtained by TruePrime™ MDA (middle blue line represents mean coverage).
Typical DNA yields from a TruePrime™ WGA kit reaction are about 5 µg per 50 µL reaction when starting with 1 ng of DNA. Yields and kinetics will vary if crude or un-quantified samples are amplified. Reactions without input DNA (no template controls) do not produce any amplification product during 3 hour reactions. Mean product length is greater than 10 kb. Store amplified DNA at 4°C for short-term or at -20°C for long-term storage.
Each batch of TruePrime WGA Kits is tested against predetermined specifications to ensure consistent product quality. Enzymes used in the kit have been tested separately to ensure adherence to specifications.
Shipping and Storage:
Kits are shipped on dry ice. Upon receipt, the kit should be stored immediately at -20°C in a non-frost-free (constant-temperature) freezer. When stored under these conditions and handled correctly, the products can be kept at least six months after shipping without showing any reduction in performance. For longer periods of time, store the kit at -80°C.