Low Binding DNA Products

Sensitive applications such as Next Generation Sequencing (NGS) sample preparation applications demand the highest quality consumables. Low binding polypropylene tubes from 4titude allow for multiple incubation and transfer steps without sample loss

Polypropylene (PP) is the best plastic material for PCR tubes as it is chemically inert, resistant to solvents, and well suited for injection moulding - allowing for production of thin-walled tubes for optimum PCR results.

DNA has been shown to bind to PP tubes especially at high ionic strength, despite the very hydrophilic nature of this material. Different PP polymers are used for the production of PCR consumables and as they differ in their characteristics including surface charges, they consequently bind DNA in varying amounts.

DNA binding to PP surfaces has typically only been an issue for reaction tubes and storage vessels but not for PCR/qPCR tubes. DNA sticking to tube walls is either released during denaturation steps and/or remains accessible for amplification.

Nevertheless, due to a progressing volume miniaturisation and with new technologies such as Next Generation Sequencing (NGS), PCR/qPCR tubes are more and more also recommended for other applications which may require ultra-low DNA binding.

• Low binding feature results from selected low-bind polymers, no coating is used to achieve the binding characteristics - No contamination or modification of the samples

• Tested under a broad temperature range - Maximum DNA recovery after low temperature storage and high temperature incubation

• No DNA loss during incubation steps and sample transfer in NGS sample prep and library construction - Ideally suited for sensitive applications with ultra-low DNA input

Experimental Data

Fig 1, Binding of linear DNA to different PP polymers - Experimental set-up

Experimental set-up

A tenfold DNA dilution series was created containing a linear 1176 bp product (digested from plasmid ACTA1, TA cloned into the pGemT vector) resulting in DNA concentrations of 10 ng/µl, 1 ng/µl, 0.1 ng/µl, and 0.010 ng/µl.

50 µl of each DNA concentration were applied to a PCR plate and incubated for 30 minutes at 37°C. The DNA was afterwards transferred to the next row of the PCR plate for additional 30 minute incubation. This procedure was repeated seven times so that the DNA was incubated in 8 different tubes for a total of 240 minutes. All transfer steps were performed with commercially available low-binding tips (Corning).

2 µl of each DNA concentration were subsequently subjected to qPCR analysis and compared to 2 µl of the original dilution series (total DNA input = 2 ng to 0.002 ng per qPCR run) using ACTA1 primer and probe set designed by IdT (Integrated DNA Technology).

Further experiments were performed with the 4titude low binding PP polymer. The experiment described above was repeated with identical experimental conditions using mouse genomic DNA (dissolved in 5 mM Tris-HCl pH 8.0) instead of the ACTA1 fragment testing three different incubation temperatures (4°C as a typical storage temperature for reaction mixtures, 37°C as a typical temperature for enzyme reactions, and 65°C as a typical temperature for enzyme denaturation). qPCR comparisons were performed with identical DNA concentrations using KAPA SYBR Fast qPCR Master Mix.

Figure 3 shows an exemplary standard curve taken from the 4°C incubation showing the Ct value for 4 different input concentrations for incubated samples versus controls. The results for other temperatures were comparable.