One of the main challenges for cell-free DNA analysis is the limited amount obtained from any bodily fluid, which affects the number and type of tests that can be performed. The TruePrime™ apoptotic cell-free DNA amplification kit provides the solution to this issue by exponentially amplifying cell-free DNA derived from apoptosis cell-death mechanism (160-170 bp).
Features and benefits
Excellent sensitivity – down to picograms of input material
High amplification yields
Flexibility, up to 150 ng or 50 µl of cfDNA input
Error-free amplification due to the use of high-fidelity Phi29 DNA pol
No artefacts derived from random synthetic primers
Streamlined workflow and reduced hands-on time
The TruePrime™ apoptotic cell-free DNA amplification kit uses a novel multiple displacement amplification method to achieve accurate DNA amplification from cell-free DNA obtained from plasma, serum, urine, CSF or any other bodily fluid. TthPrimPol generates primers on the hairpin-adaptors that are extended by Phi29 DNA polymerase. The strong strand displacement capacity of Phi29 DNA polymerase allows TthPrimPol to synthesize new primers on the new hairpins generated, resulting in exponential isothermal DNA amplification.
The kit combines the novel TruePrimeTM DNA amplification technology, with key novel steps of cell-free DNA pretreatment, composed of an end-repair + dA tailing reaction and ligation (Figure 1) of hairpin-adaptors (Figure 2), which enables the efficient amplification of apoptotic cell-free DNA by TruePrimeTM following the rolling circle DNA amplification method.
Figure 1. Cell-free DNA pretreatment to eliminate protruding ends, restore 5’ phosphates and 3’ hydroxyl groups, and add 3’ dA overhangs. Once the cfDNA is repaired, hairpin-adaptors are ligated to both ends of each cfDNA molecule.
Figure 2. DNA sequence and structure of the hairpin-adaptor used in the TruePrimeTM apoptotic cell-free DNA amplifi cation kit.
Amplification mechanism of the novel TruePrimeTM apoptotic cell-free DNA amplifi cation kit
TthPrimPol generates primers on the hairpin-adaptors that are extended by Phi29 DNA pol. The strong strand displacement capacity of Phi29 DNA polymerase allows TthPrimPol to synthesize new primers on the new hairpins generated, resulting in exponential isothermal DNA amplification.
Shipping and storage
The TruePrime™ Whole Cell-Free DNA Amplification Kit is shipped in dry ice. Upon receipt, the kit should be stored immediately at -20ºC in a non-frost-free (constant-temperature) freezer. If stored correctly, the product can be kept for at least 6 months after shipping without displaying any reduction in performance. For longer periods, store the kit at -80ºC. Avoid repeated free-thaw cycles if possible.