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TruePrime® Apoptotic Cell-Free DNA Amplification Kit

One of the main challenges in cell-free DNA analysis is the limited amount obtained from any bodily fluids, 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 challenge 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 artifacts 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 methods to achieve accurate DNA amplification from cell-free DNA obtained from plasma, serum, urine, CSF or any other bodily fluids. TthPrimPol generates primers on the hairpin-adaptors which are in turn 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 TruePrime® DNA amplification technology, with key novel steps of cell-free DNA pre-treatment, 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 TruePrime® following the rolling circle DNA amplification method.

TruePrime Apoptotic kit - Cell-free DNA pretreatment

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.

TruePrime Apoptotic kit - DNA sequence and structure of the hairpin-adaptor

Figure 2. DNA sequence and structure of the hairpin-adaptor used in the TruePrime® apoptotic cell-free DNA amplification kit.

Amplification mechanism of the novel TruePrime® apoptotic cell-free DNA amplification 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 primers on the new hairpins generated, resulting in exponential isothermal DNA amplification.

Amplification mechanism of the TruePrimeTM apoptotic cell-free DNA amplification kitShipping 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.

 

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Expected amplification yields

48 cancer patients were recruited and 10 mls of blood were extracted using Streck Cell-free DNA BCT® tubes. 3 mls of plasma were immediately isolated through a double-spin centrifugation protocol to avoid genomic DNA contamination from nucleated blood cells. Cell-free DNA was purified from 1ml of plasma using Qiagen QIAmp® Circulating Nucleic Acid Kit. Cell-free DNA was quantified using QubitTM. 1 ng of each cell-free DNA sample was subjected to the TruePrimeTM apoptotic cell-free DNA amplification workflow. The figure below shows the DNA amplification yields obtained in each case. All cell-free DNA samples were efficiently amplified, producing enough DNA for any subsequent analysis or technique.

TruePrime apoptotic kit expected amplification yields

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