A magnetic bead-based method for concentrating DNA from human urine for downstream detection.

Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×10(3) to 5×10(8) copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×10(6), 14×10(6), and 8×10(6) copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR

Genetic and Maternal Effects on Valproic Acid Teratogenesis in C57BL/6J and DBA/2J Mice

Valproic acid (VPA) is used worldwide to treat epilepsy, migraine headaches, and bipolar disorder. However, VPA is teratogenic and in utero exposure can lead to congenital malformations. Using inbred C57BL/6J (B6) and DBA/2J (D2) mice, we asked whether genetic variation could play a role in susceptibility to VPA teratogenesis. Whereas B6 fetuses were more susceptible than D2 fetuses to digit and vertebral malformations, D2 fetuses were more susceptible to rib malformations. In a reciprocal cross between B6 and D2, genetically identical F1 mice carried in a B6 mother had a greater percentage of vertebral malformations following prenatal VPA exposure than F1 mice carried in a D2 mother. This reciprocal F1 difference is known as a maternal effect and shows that maternal genotype/uterine environment is an important mediator of VPA teratogenecity. VPA is a histone deacetylase inhibitor, and it is possible that the differential teratogenesis in B6 and D2 is because of strain differences in histone acetylation. We observed strain differences in acetylation of histones H3 and H4 in both embryo and placenta following in utero VPA exposure, but additional studies are needed to determine the significance of these changes in mediating teratogenesis. Our results provide additional support that genetic factors, both maternal and fetal, play a role in VPA teratogenesis. Lines of mice derived from B6 and D2 will be a useful model for elucidating the genetic architecture underlying susceptibility to VPA teratogenesis

The percentage of DNA recovered from 1 mL urine samples increases as the concentration of magnetic beads increases to 6×10⁸ beads/mL (mean ± s.d., n = 3).

<p>The percentage of DNA recovered from 1 mL urine samples increases as the concentration of magnetic beads increases to 6×10⁸ beads/mL (mean ± s.d., n = 3).</p

There was no significant change in DNA recovery with increased adsorption (white circles) or elution (black circles) times (mean ± s.d., n = 3).

<p>In each experiment, the adsorption time was held at 30 s while the elution time was varied (black circles), and the elution time was held at 30 s while the adsorption time was varied (white circles). To more clearly illustrate the error bars on each data point, the adsorption data was plotted two seconds to the right at each time point.</p

DNA recovery is significantly increased with increasing DNA sequence length (mean ± s.d., n = 3).

<p>* denotes statistically different than 75 bp sequence.</p

The LOD of the PCR reaction is shown at 8800 copies (dotted line).

<p>The lowest initial concentration of IS6110 DNA detectable at the LOD was determined to be 77 copies/µL at the point at which this intersects the PCR-concentration curve (red circle) (mean ± s.d., n = 3).</p

The DNA recovery for the magnetic bead-based extraction method and a commercially available laboratory-based kit are comparable.

<p>Though both are significantly higher than the unextracted sample, which is undetectable by PCR, there is no statistical difference between the two extraction methods (mean ± s.d., n = 9); * denotes statistically higher recovery than unextracted sample.</p

The final concentration of IS6110 DNA in the eluate following magnetic bead-based extraction of 3 and 5 mL spiked urine samples is significantly higher than the IS6110 DNA concentration in the eluate following the extraction of 1 mL samples (mean ± s.d., n = 3); * denotes statistically different from 1 mL sample.

<p>The final concentration of IS6110 DNA in the eluate following magnetic bead-based extraction of 3 and 5 mL spiked urine samples is significantly higher than the IS6110 DNA concentration in the eluate following the extraction of 1 mL samples (mean ± s.d., n = 3); * denotes statistically different from 1 mL sample.</p

There is no significant difference in DNA recovery using the lyophilized urine collection pipette stored for 0, 4, 8, or 12 weeks (black circles) compared to freshly prepared adsorption reagents (white circle) (mean ± s.d., n = 3,).

<p>There is no significant difference in DNA recovery using the lyophilized urine collection pipette stored for 0, 4, 8, or 12 weeks (black circles) compared to freshly prepared adsorption reagents (white circle) (mean ± s.d., n = 3,).</p