WO2013067069A2 - Dual digestion method for high yield nucleic acid recovery - Google Patents

Abstract

Methods and kits for isolating nucleic acids from a sample are provided.

Description

TITLE

Dual Digestion Method for High Yield Nucleic Acid Recovery

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial No.

61/553,842, filed on October 31, 2011, which is incorporated herein by reference in its entirety.

FIELD

Methods and kits for isolating nucleic acids from a sample are provided.

BACKGROUND

Traditionally, nucleic acid extraction is based on lysing and digesting a sample in alkaline to neutral conditions with alkaline or neutral loving enzymes. This is primarily due to degradation of DNA and RNA under acid conditions, especially at pH 3 or less. Often higher temperatures are used to accelerate the digestion, but this also risks accelerating chemical degradation from loss of purine nucleotides. Most reactions are accelerated at higher temps including depurination. On the other hand, mild acidic digestion with acid loving enzymes at low or room temperature tends to take a long time and may result in incomplete digestion of proteins, especially in protein rich samples.

This may result in losses and lower recovery of nucleic acids. Some nucleic acids bind to proteins and protein histones which, if incompletely broken down, may be washed away with the supernatant. This is especially problematic in samples with high protein content and low nucleic acid content.

Most commercially available nucleic acid isolation protocols tend to use only one protease, usually proteinase K. A one protease system may work well in samples in which nucleic acids represent a sizeable fraction of the total sample mass, but a one protease system is less efficient when the amount of protein in a sample greatly exceeds the amount of nucleic acids in the sample. SUMMARY

A method for isolating nucleic acids from sources in which the amount of protein greatly exceeds the amount of nucleic acids is provided. Samples may include, but are not limited to blood serum, plasma, hair, nails, feces, and bird feathers.

In one embodiment, recovery of nucleic acids is achieved by use of an acid based digestion, using pepsin or other proteases active at low pH.

In one embodiment, recovery of nucleic acids is achieved by use of a two protease digestion with pepsin or other proteases active at low pH used as a second protease. The sample is taken from the first alkali to neutral digest in a choatrope to the second digest by adjusting the pH to about pH 4 to about pH 5. Pepsin has been shown to be active in urea at pH 4. Additionally, the conditions of about pH 4 and about room temperature disfavor the depurination reaction, but favor the binding of phosphate groups on the nucleic acids to the metal oxide groups on the capture matrix.

In general, a first digestion can be at any neutral to alkali pH in a chaotrope and a reducing agent that will inhibit nucleases and denature highly keratinized or other tissue in which the nucleic acids are embedded in an intractable protein matrix.

In another embodiment, the digestion can be carried out under any conditions and with any protease such that the pH is below the isoelectric point of the metal oxide binding matrix and the pH and temperature are low enough so the depurination reaction is not favored, generally from about pH 3.5 to about pH 7.

BRIEF DESCRIPTION OF FIGURES

Figure 1 is a gel of DNA isolated in Example 1.

Figure 2 is a gel of GSTlp PCR products from DNA isolated in Example 1. Figure 3 is a gel of GSTlp PCR products from DNA isolated in Example 5.

Figure 4 shows UV spectra of nucleic acids isolated in Example 6.

Figure 5 is a gel of DNA isolated in Example 6.

Figure 6 is a gel of GADPH PCR products from DNA isolated in Example 6. Figure 7 contains UV spectra of nucleic acids isolated from pigeon feathers in Example 7.

Figure 8 contains the UV spectra of nucleic acids isolated from hair and ground beef in Example 8.

Figure 9 contains the UV spectra of nucleic acids isolated from pigeon feces in Example 9.

Figure 10 contains the UV spectra of nucleic acids isolated from Example 13.

Figure 11 shows UV absorption as a function of digestion time for nucleic acids isolated from Example 13.

Figure 12 shows nucleic acid concentration as a function of digestion time for nucleic acids isolated from Example 13.

Figure 13 is a gel of DNA isolated in Example 13.

Figure 14 is a gel of GADPH PCR products from DNA isolated in Example 13. Figure 15 is a gel of DNA isolated in Example 14.

Figure 16 is a gel of GADPH PCR products from DNA isolated in Example 14.

Figure 17 is a gel of DNA isolated in Example 15.

Figure 18 is a gel of GADPH PCR products from DNA isolated in Example 15. Figure 19 is a gel of DNA isolated in Example 16.

Figure 20 is a gel of GADPH PCR products from DNA isolated in Example 16. Figure 21 is a gel of DNA isolated from various nanoparticles in Example 17.

Figure 22 is a gel of DNA isolated in Example 18. Figure 22 A is an image taken early during electrophoresis, and Figure 22B is an image taken later during

electrophoresis.

Figure 23 is a gel of GADPH PCR products from DNA isolated in Example 18. Figure 24 shows UV absorbance spectra of the nucleic acids isolated using the magnetic beads described in Example 19.

Figure 25 contains graphs of UV absorbance or nucleic acids isolated in Example 19. Figure 25A is shows the approximate RNA concentration. Figure 25B shows the 260/230 ratio. Figure 25 C shows the 260/280 nm ratio.

Figure 26 is a graphic representation of the concentrations of nucleic acid isolated in Example 19. In Figure 26 A, DNA concentrations as measured by PicoGreen are shown. In Figure 26B, RNA concentrations as measured by RiboGreen are shown.

Figure 27 is a gel of DNA isolated in Example 19.

Figure 28 is a gel of GADPH PCR products from DNA isolated in Example 19. Figure 29 is a graphic representation of the band densities from Scion Image for the DNA isolated in Example 19 (Figure 27) for the and GADPH PCR products (Figure 28) from Example 19.

Figure 30 is a gel of DNA isolated in Example 20. Figure 31 is a gel of GADPH PCR products from DNA isolated in Example 20.

Figure 32 is a gel of DNA isolated in in Example 21. Figure 32 A shows DNA and RNA migrating faster than the 50 base pair standard. Figure 32B shows DNA greater than 10,000 base pairs.

Figure 33 is a gel of GADPH PCR products from DNA isolated in Example 21.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A method for isolating nucleic acids from a sample are provided. By finding an appropriate balance of acidic pH (below 3) and temperatures, it is possible to neutralize the nucleases and to achieve a more complete digestion of the proteins using acid loving enzymes such as pepsin. Acid loving enzymes are very aggressive in these conditions and result in a quick and thorough digestion. Nucleic acid degradation due to acid conditions is minimized at about pH4, and degradation due to nuclease activity is reduced or eliminated at these conditions resulting in higher yields of nucleic acids.

There are pH and temperature "tipping points" for the deleterious reaction of depurination. The first tipping point is pH, around pH 3. Above pH 3, depurination proceeds much more slowly. The second tipping point is temperature, around 30°C. Below this temperature, depurination proceeds much more slowly.

Pepsin has optimal activity at pH 2, but it can become very active at pH 4 to pH5 in the presence of a chaotrope such as urea. Even though pepsin has an optimal activity at 37°C to 60°C, it remains active at 25°C, a temperature at which depurination is below the tipping point. Due to the economy of pepsin, the decrease in the activity of pepsin may be compensated for my adding more enzyme. By combining and taking advantage of these characteristics, it is possible to balance the various factors involved and develop a method of isolating nucleic acids from sources that traditionally produce very poor yields.

Various digestion processes, based on an acid digestion and extraction, can be optimized for various samples and target nucleic acids.

In one embodiment, an acidic condition digestion using acid loving enzymes for nucleic acid extraction and purification is provided. This type of digest results in better yields than alkaline conditions for samples such as rich in protein. Phosphate containing compounds, such as nucleic acids, can interact with metal oxide matrixes. This nucleic acid capture can be combined with a mild acidic digest. Traditional methods rely on making nucleic acids less soluble by the addition of alcohols. One disadvantage of this method is that the addition of alcohols also makes partially digested proteins less soluble. The digestion methods described take advantage of finding a balance between pH and temperature where proteins are adequately digested and nucleic acids do not undergo excessive depurination.

In one embodiment, a dual digestion is provided, first under alkaline conditions with alkaline loving enzymes, and then under acid conditions with acid loving enzymes. This results in a more complete digestion of proteins and the release of the nucleic acids.

The dual digest can be done with or without alcohol precipitation steps. In the dual digestion with alcohol precipitation steps, the alcohol precipitation after the alkaline digest serves to remove the chaotropic agent, such as urea or guanidine HC1. Other chaotropic agents include lithium perchlorate, lithium acetate, magnesium chloride, and thiourea.

The dual digest can also be done without an alcohol precipitation step after the alkaline digest. This takes advantage of the fact that pepsin may be very active in urea if the pH is in the 4-5 range. In this process, no precipitation step is inserted after the allkiline digestion. Instead, the partly digested sample is titrated to an acidic condition, and the acid loving enzyme is introduced. This method is most suited for automation.

The digestion methods described can be used with any downstream extraction or purification methods, for example magnetic beads, alcohol precipitation, filters, spin columns, other methods of chromatography, etc.

Ending the digestion in these acidic conditions allows for the utilization of the direct affinities of metallic and ceramic materials to bind nucleic acids at these specific pH ranges and release them by shifting those conditions. Furthermore, selecting specific metals in the binding matrix can be utilized to select for larger or smaller nucleic acid fragments, or to selectively isolate DNA or RNA.

Metallic and ceramic beads, magnetic beads, meshes, filters, etc. can all be used. One preferred embodiment is metallic and ceramic nanoparticles and magnetic versions of the same. This allows for the capture media to be dispersed and separated when the target nucleic acid is released without trapping any of the nucleic acid and reducing systemic losses.

A method of isolating nucleic acids from a sample is provided. In one embodiment, the method includes a dual digest and purification. The sample is first treated with an enzymatic protease digestion in alkaline conditions with neutral or base- loving enzymes such as Savinase or ProteaseK in the presence of reducing agents, such as beta-ME. Then the sample is optionally subjected to an alcohol precipitation to drive down the nucleic acids, lipids, and incompletely digested proteins. A second digestion is carried out under acidic conditions, at a pH of about 4 to about 5, with enzymes that work well in acids, such as pepsin or other acid proteases. Then, utilizing the affinity of ceramics for nucleic acids under these acid conditions, nucleic acids can be pulled out of the digested solution directly without an additional precipitation step.

It is thought that the alkaline digestion with the reducing agents breaks down DNase and RNase that may be present in the sample, thereby reducing further degradation of the DNA or RNA. The alkaline digestion also starts to break down the proteins present, but some of the nucleic acids are likely still bound to these proteins. In a traditional nucleic acid extraction in which the proteins are precipitated out at this point, any nucleic acids still bound to those proteins would also be lost in the precipitation. There may also be a significant amount of DNA that is not completely released from the protein histones which are not completely broken down in the first digestion. In lipid rich samples such as blood, serum, or plasma nucleic acids may also be trapped by lipids which are not completely broken down.

In the present method, the inefficiencies present in a traditional alkaline digestion are utilized so that the nucleic acids bound to proteins and lipids are precipitated with the proteins and lipids rather than being lost in the isolation procedure.

A second digestion is carried out under acid conditions with an enzyme or enzymes such as pepsin. This digestion further breaks down proteins into peptides and allows the release of nucleic acids that may be bound to the proteins. The result is that much greater yields of nucleic acids are recovered than found in traditional isolation methods.

In a preferred embodiment, there is no additional alcohol precipitation step after the alkaline digest. In this case, the chaotropic agent may enhance the activity of the acid digest.

There is no additional precipitation step. The nucleic acids are pulled out of solution based on the affinity of nucleic acids to the nanoparticles in the pH range of about pH 4 to about pH 5. At this pH, pepsin is still active, and there is a balance between activating the affinity of nucleic acids for the nanoparticles, yet minimizing the degradation of nucleic acids. The elution or release of nucleic acids process is accomplished by changing the pH.

The methods described are optimized for recovery of nucleic acids from high protein and/or lipid containing samples with low nucleic acid content. Such samples may include, but are not limited to, the following: cell free DNA/RNA from serum or plasma; hair; feathers; FFPE samples; nails; muscle and sinew tissue; fossil and archaic tissue samples; and bone. These methods are also useful for isolating nucleic acids from any small sample where efficient recovery of nucleic acids is required. These isolation methods may prove extremely valuable in applications including, but not limited to, the following: cancer screenings; medical diagnostics; needle biopsies; laser capture microdissection; and forensics.

The methods provided can enhance any nucleic acid extraction in which recovery of even very small or very degraded nucleic acid fragments may be required. There is a growing need for nucleic acid isolation methods useful for isolating nucleic acid from small, rare, dilute, or difficult to recover samples.

In one embodiment, the isolation method is generally carried out as follows. A blood sample from a patient is centrifuged to separate plasma from cells. The plasma is withdrawn and placed in a test tube containing Savinase. Beta-mercaptoethanol or a similar reducing agent is added. After the sample is digested for about 30 to 60 minutes at 55°C, nanoparticles are added in the volume of O.Olx the volume of plasma. The sample is agitated or vortexed to disperse the nanoparticles. Nucleic acids and partially digested protein are precipitated onto the nanoparticles by adding at least 2 volumes of 70% isopropanol (2PrOH). The pellet containing nanoparticles, nucleic acids, proteins, and some lipid is resuspended in a 2 mg/mL solution of pepsin also containing 5 mM HCl such that there is 10 mg pepsin for every 1 mL of original plasma. Additional 0.1 N HCl is added in order to adjust the pH to about 3.7 to about 4.0. The remaining protein is digested for about 30 minutes to one hour at 25° to 37 °C. The nucleic acids and nanoparticles are collected by centrifuging at 2000g for 5 minutes. Residual HCl is rinsed from the nanoparticles and nucleic acids with 5 mL 70% isopropanol. The supernatant is discarded. Residual isopropanol is evaporated by heating at 54°C. Nucleic acids may be eluted with, for example, 10 mM Tris pH 8, 10 mM Na₂HP0₄, or 10 mM Na₂B₄0₇ pH 9, depending on the downstream application. There are several advantages of this method of isolating nucleic acids. The first digestion is thought to disable nucleases rapidly because it is carried out at physiological pH. The second digest is carried out at an acidic pH with a proteinase such as pepsin that has activity at the same pH that allows phosphate groups on nucleic acids to bind to metal oxide groups on nanoparticles.

The present method appears to yield cell free DNA as well as very large quantities of short strands of RNA that may include both miRNA and mRNA. Due to the excess of RNA, it is possible to proceed with downstream application such as miRNA microarray analysis without removal of other nucleic acids or additional concentration steps. For downstream analysis of cell free DNA, however, it may be preferable to further purify the DNA, depending on the application.

While nanoparticles are used in various preferred embodiments, other

nano structures as well as larger structures may be used. The capture media may be, for example, particles, tubes, fibers, rods, filaments, or spheres. Structures on a micron scale or larger may also be used. Additional nondispersive structures such as wires, screen, or mesh may also be used. Additionally, useful ceramics may be embedded into other types of media such as paper. Preferred embodiments utilize passivated ceramic metal oxide surfaces for the capture media. EXAMPLES

Example 1 Suboptimal temperature and pH do not affect DNA integrity and ability to amplify sequences in a multiplex PCR system

Partially purified urine cell free DNA was treated to isolate plasma cell free DNA. The procedure was carried out as follows: Aliquoted 2x lmL of urine RNA/DNA. This particular preparation was 18 uM RNA as measured by RiboGreen (Molecular Probes Eugene, OR) and 2 uM DNA as measured by PicoGreen (Molecular Probes, Eugene, OR). Added 20 uL of Zr0₂-B₄0₇ nanoparticles and 50 uL of 10 M LiCl. (LiCl is not added in the plasma cell free DNA (cfDNA) prep because sufficient salts are already present in the plasma.) Precipitated by adding 1 mL 100% 2PrOH. Vortexed. Waited 5 minutes. Centrifuged at 8000 g for 2 minutes. Retained the pellet. Tared out 46 mg of Amresco pepsin, ProPure M142-250g lot 0019B466. Transferred to 15 mL tube. Added 0.4 mL 10 mM HC1. Brought volume to 4 mL with deionized water. Added 2 mL of this solution to each pellet. Final pH 5 as measured by Whatman 0-14 pH paper. One tube was kept at room temperature (approximately 25 °C) and the other kept at approximately 35°C. At 15, 30, 60, and 120 minute time points, 250 uL aliquots were removed and pipetted up and down to get nanoparticles dispersed. Transferred 250 uL aliquots to 500 uL polypropylene tubes. Centrifuged for 2 minutes. Rinsed captured DNA on the nanoparticle pellet with 250 uL 70% 2PrOH. (#2) Transferred supernatants (#3) to 500 uL polypropylene tubes and added 6 uL Zr02-B₄07 nanoparticles and 12 uL 10 M LiCl. Precipitated by added 250 uL 100% 2PrOH. Waited 5minutes. Centrifuged for 2 minutes. Pellets were allowed to dry. Nucleic acids were eluted into 50 mL 12 mM Na₂HP0₄ pH 8.0. Samples were centrifuged for 2 minutes.

The following GST 6-plex PCR primers were used at 0.4 uM. The total reaction volume was 20 uL with 2 uL sample.

SEQ ID NO: 1 - GSTP1 C-RP 5 ' - etc aaa agg ctt cag ttg cc - 3 '

SEQ ID NO:2 - GSTPlC-FP-167 5' - gga gca age aga gga gaa tc - 3' SEQ ID NO:3 - GSTPlC-FP-244 5' - aag gat gga cag gca gaa tg - 3'

SEQ ID NO:4 - GSTPlC-FP-330 5' - ggc tgt gtc tga atg tga gg - 3'

SEQ ID NO:5 - GSTPlC-FP-398 5' - cga agg cct tga acc cac t - 3'

SEQ ID NO:6 - GSTP1 C-FP-473 5 ' - cgt gtg tgt gtg tac get tg - 3 '

SEQ ID NO:7 - GSTPlC-FP-551 5' - cag aca cag age aca ttt gg - 3'

The thermocyler program for the PCR reaction was done as follows: step 1 : hot start at 94°C for 4 minutes; step 2: denature at 94°C for 1 minute; step 3: anneal at 68°C for 1 minute (x34); step 4: extend at 72°C for 1 minute; step 5: final extension at 72°C for 7 minutes; step 6: hold at 15 °C.

PCR products (5 uL) as well as raw DNA (10 uL) were resolved on a double tiered 2.2% Lonza Flashgel (Rockland, ME). Images during electrophoresis were captured with a Flashgel camera also from Lonza. Raw DNA samples were allowed to evaporate to 6-7 uL on parafilm. Images were imported into Adobe Photoshop 7.0. The following manipulations were performed: The image was inverted in the Image

Adjustment tool bar. Levels, also in the Image Adjustment toolbar, were adjusted such as to best to visualize differences in the six PCR products. The PCR negative and positive (starting urine DNA sample) control as well as raw DNA from the starting sample were run on a separate gel. Images were imported into Scion Image. The GelPlot2 macro was used to quantitate band intensities. Loading for the first set was as follows: lane 0 - Sigma DNA ladder; lane 1 - 15 minutes at 25 °C, no alcohol precipitation (no R-OH ppt); lane 2 - 30 minutes at 25 °C (no R-OH ppt); lane 3 - 60 minutes at 25°C (no R-OH ppt); lane 4 - 120 minutes at 25° (no R-OH ppt); lane 5 - 15 minutes at 25°C with alcohol precipitation (with R-OH ppt); lane 6 - 30 minutes at 25 °C (with R-OH ppt); lane 7 - 60 minutes at 25 °C (with R-OH ppt); lane 8 - 120 minutes at 25°C (with R-OH ppt); lane 9 - 15 minutes at 35°C (no R-OH ppt); lane 10 - 30 minutes at 35°C (no R-OH ppt); lane 11 - 60 minutes at 35°C (no R- OH ppt); lane 12 - 120 minutes at 35°C (no R-OH ppt); lane 13 - 15 minutes at 35°C (with R-OH ppt); lane 14 - 30 minutes at 35°C (with R-OH ppt); lane 15 - 60 minutes at 35°C (with R-OH ppt); lane 16 - 120 minutes at 35°C (with R-OH ppt); lane 17 - negative control; lane 18 - urine.

Figure 1 contains raw DNA. There are four major bands in the starting DNA at approximately 10,000 base pairs, slightly greater than 500 base pairs, 100-300 base pairs, and less than 50 base pairs. Results from the pH 4-5 pepsin digestion show the following characteristics. The DNA at greater than 10,000 base pairs is enriched compared to the starting material (lane 18). No time-dependent degradation of DNA is seen (lanes 1-4 and 9-12). Additionally, most of the DNA is captured by the nanoparticles at pH 4-5 (fraction #2, no alcohol precipitation) without the use of additional salts and alcohol (fraction #3, with alcohol precipitation.

In Figure 2, all six GSTlp PCR products are present in samples heated both at

25°C and at 37°C for 120 minutes, or two hours. Qualitatively, the amplicons from samples digested with pepsin (fraction #2 that was captured by nanoparticles without alcohol) appear cleaner than those from the starting material. Example 2 Protocol used to isolate cell free DNA (cfDNA) from pooled human plasma.

Frozen pooled normal human plasma (Bioreclammation, BRH415419) was chipped away with a metal spatula and quickly transferred to a 15 mL polypropylene conical tube. Stopped chipping away when the apparent volume was close to 1.5 to 2 mL. Before the plasma was completely thawed, 25 uL of 16U/g Savinase (Novazymes) and 25 uL β-mercaptoethanol (Sigma) were added. The material was digested for 1 hour at 54 °C in a heat block. The digest was split between two 15 mL conical tubes. Added 15 uL of phosphate passivated Ce0₂ (CP) as well as borate passivated Ce0₂ (CB). Precipitated undigested protein, DNA, and particles by adding 70% 2PrOH to the 11 mL mark. Centrifuged at 2000g for 5 minutes. The supernatant (fraction #1) was retained long enough to measure the UV absorbance and then discarded. The UV absorbance at 280 nm indicates the amount of protein being digested and removed. 15 mg porcine stomach pepsin

(Amresco) was dissolved in 10 mL 10 mM HC1. The pellet, containing DNA, nanoparticles, and undigested protein, was resuspended in this mixture. The digests were split two ways and digested for 1.5 hours at 54°C, 2.5 mL in each of four 15 mL tubes. After the pepsin digestion, the mixture was centrifuged at 2000g for 10. Pellets (fraction #2) were rinsed once with 5 mL 70% 2PrOH to remove residual HC1. The supernatants were transferred to new 15 mL polypropylene conical tubes. 15 mL of each variety of particle was added to the supernatants of the pepsin digests (fraction #3). Added 70% 2PrOH to the 15 mL mark. The final concentration was about 47%. Pellets were rinsed with 5 mL 70% 2PrOH to remove excess HC1. All pellets evaporated to dryness in a 55°C oven. Pellets were eluted with 0.5 mL 10 mM Tris pH 8.0.

Example 3 Isolation of nucleic acids from plasma comparing pepsin digestion times

A 4 mL chunk of frozen pooled normal human plasma was digested with Savinase in one 50 mL tube. 80 uL of Ce0₂-B4C>7 nanoparticles were added. The same proportion of 70% 2PrOH was used for the precipitation. Centrifugation was performed at 730g.

The pH was measured at discrete steps as the pellet was prepared for pepsin digestion.

The final pepsin digest volume was 5x the original plasma volume. At the designated time points, 5 mL aliquots were taken from the 50 mL polypropylene conical tubes and processed as in Example 2. Nucleic acids from 1 mL plasma were eluted from nanoparticles with 0.5 mL lOmM Tris pH 8, a 2x concentration. RNA and DNA were measured with RiboGreen and PicoGreen according to the manufacturers' protocols using miR-155 and a Hind!II digest of lambda DNA as standards. Example 4 [DNA] and [RNA] of pepsin digestion times

As shown in Table 1, the pH 2.9 digest at 30 minutes yielded less DNA than longer digests and more RNA than longer digests. Very little extra nucleic acids were captured in Fraction #3, see Example 2. The "ug/mL" nucleic acid numbers reflect the concentration of the eluted material which is significant for downstream applications. DNA and RNA in the original plasma, "ug/mL plasma," indicates improvements over prior art.

Table 1

Table 2 shows results where nucleic acids were eluted with 0.5 mL lOmM Tris pH 8 nanoparticles and then with 12 mM Na₂HP0₄. Approximately 10% to 140% more RNA was obtained with the second elution. Essentially no more than 25% more DNA was obtained with the second 12 mM Na₂HP0₄ elution. The yields of DNA per 1 mL plasma in the preparations used were about 1 ug/mL.

Table 2

Example 5 Comparing pepsin digestion time, PCR, and input DNA

In this example, PCR was performed with the GST IP multiplex primers on the #2 samples from Example 4. 5 uL of samples were used in 20 uL PCR reaction volumes. The genomic DNA positive control did not perform well in PCR and/or it was not resolved well on the 2.2% agarose Lonza gel.

Figure 3 is a gel of the PCR products. The lanes of the gel were loaded as follows: lane 0 - Sigma direct load wide range; lane 1 - negative control; lane 2 - Roche gDNA positive control; lane 3 - 25Febll #2 30 minutes; lane 4 - 25Febll #2 60 minutes; lane 5 - 25Febll #2 90 minutes; lane 6 - 25Febll #2 120 minutes; lane 7 - 28Febl l #2 30 minutes; lane 8 - 28Febll #2 60 minutes; lane 9 - 28Febll #2 90 minutes; lane 10 - 28Febl l #2 120 minutes, little DNA; lane 11 - 28Febll #2 30 minutes, Na₂HP0₄ elute; lane 12 - 28Febll #2 60 minutes, Na₂HP0₄ elute; lane 13 - 28Febl l #2 90 minutes, Na₂HP0₄ elute; lane 14 - 28Febll #2 120 minutes, Na₂HP0₄ elute; lane 15 - 25Febll #3 30 minutes; lane 16 - 28Febl l #3 30 minutes, Na₂HP0₄ elute.

Example 6: Comparing two nanoparticles and oxyanion passivations

In this example, experiments were carried out to determine whether ceria (Ce0₂) or zirconia (Zr0₂) was a more effective nanoparticle and if borate (B₄0?) or phosphate (P0₄) was a more effective passivating agent. The two way ANOVA design was used to cover the possibility that there may be an interaction between the nanoparticle base and the type of passivation.

Figure 4 shows DNA/RNA samples eluted from the nanoparticles with 12 mM

Na₂HP0₄ that were diluted lOx in the same solution in order to measure the UV absorbance. Note the absorbance maxima at 220 to 225nm. The 260/230 nm ratio is one common index of DNA/RNA purity. Slightly fragmented nanoparticles contribute to the absorbance at 220nm. The 260/280nm ratio is an index of protein contamination because aromatic amino acid groups absorb at 280nm.

UV absorption data in Table 3 shows the 260/280 ratios were greater for ceria nanoparticles than they were for zirconia nanoparticles (p<0.05). There was no significant difference in the nanoparticle types for the 260/230 nm ratio. The term "[DNA]" refers to what the concentration of DNA would be if the entirety of what absorbs at 260 nm were DNA. The yield of this material was greater for ceria than for zirconia (p<0.01). Borate passivation seemed to result in a slightly greater yield (p<0.05).

Table 3

DNA and RNA concentrations were quantitated using PicoGreen and RiboGreen (Molecular Probes, Eugene OR). Unlike UV absorbance values, there were no significant differences in the four nanoparticle types for DNA and RNA yield. Note that the combined DNA and RNA yields fall far short of what would be predicted by the absorbance at 260nm. This absorbance at 260nm may come from fragments of nucleic acids too small to bind to PicoGreen or RiboGreen or from a blood component such as ADP.

Table 4 PicoGreen and RiboGreen DNA and RNA concentrations

PCR was performed on the plasma cell free DNA (cfDNA) from this example using glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) primers. The GAPDH PCR protocol was as follows: start at 95°C for 4 minutes; denature at 95°C for 30 seconds; anneal at 60°C for 1 minute (34x); extension at 72°C for 1.5 minutes. The primers used were:

SEQ ID NO:8 - GAPDH forward primer 5'-ACC ACA GTC CAT GCC ATCAC-3' SEQ ID NO:9 - GAPDH reverse primer 5'-TCC ACC ACC CTG TTG CTG TA-3' (See, Liu MY, et al. , (2006) Inducible platelet-derived growth factor D-chain expression by angiotensin II and hydrogen peroxide involves transcriptional regulation by Ets-1 and Spl, Blood, 107(6):2322-9. )

Raw DNA and RNA samples were resolved on a 2.2% Lonza DNA gel which can also be used for RNA. The Lonza dye in the DNA gels will also bind to RNA, but with less affinity. The levels were adjusted in Adobe Photoshop 7.0 in order to visualize nucleic acids other than the less than 50 base pair material. The white marks above the 500 base pair marker are the negative images from the bromophenol blue dye. The lanes of the gel in Figure 5 are as follows: lane 0 - Sigma DNA ladder; lane 1 - 13Aprl la Zr0₂-P0₄; lane 2 - 13 Aprl la Zr0₂-B₄0₇; lane 3 - 13Aprl la Ce0₂-P0₄; lane 4 -

13Aprl la Ce0₂-B₄0₇; lane 5 - 13Aprl lb Zr0₂-P0₄; lane 6 - 13Aprl lb Zr0₂-B₄0₇; lane 7 - 13Aprl lb Ce0₂-P0₄; lane 8 - 13Aprl lb Ce0₂-B₄0₇; lane 9 - 13 Aprl lc Zr0₂- P0₄; lane 10 - 14Aprl lc Zr0₂-B₄0₇; lane 11 - 14Aprl lc Ce0₂-P0₄; lane 12 - 14Aprl lc Ce0₂-B₄0₇; lane 13 - 14Aprl ld Zr0₂-P0₄; lane 14 - 14Aprl ld Zr0₂-B₄0₇; lane 15 - 14Aprl Id Ce0₂-P0₄; lane 16 - 14Aprl Id Ce0₂-B₄0₇. Figure 6 shows the PCR products. The lanes in Figure 6 are the same as those in Figure 5 with the additioan of lane 17 - water blank; and lane 18 - 1 uL Roche gDNA. The expected size of the GADPH amplicon is 451 base pairs. The image of the PCR gel was imported into Scion image. The Gelplot2 macro was used to quantitate the 451 base pair bands. There is no significant difference between the four types of nanoparticles tested with respect to the 451 base pair G3PDH amplicon intensity, as shown in Table 5.

Table 5 Band Intensity using Scion Image

Example 7 : Nucleic acid isolation from pigeon feathers

Two pigeon feathers were used, and the feathers were separated from the shafts. One was 60mg, the other was 80mg. 500 uL digestion solution was added. A digestion was carried out with 9 uL beta-mercaptoethanol (β-ΜΕ) and 50 uL of Savinase. After about 1 hour digestion at 56°C, 60 uL Ce02-B₄07 particles were added. Precipitation was done by adding 0.75 mL 100% 2PrOH. Digested for 1 hour at 37°C with 1 mL 10 mg/ml Amresco pepsin in 5 mM HC1, pH 4 after mixing with pellets. A second dose of 10 mg/mL pepsin in 5 mM HC1 was added. pH was 3 or below for about 2minutes. Quickly rinsed with 1 mL 70% 2-PrOH and twice with 1 mL deionized water. The pellets were still brown, thought to be due to melanin, a browning agent. Pellets were rinsed once with 1 mL 10 mM Tris pH 8.0. Absorbance data is shown in Figure 7. Example 8 Nucleic acid isolation from ground beef and hair

264 mg of fatty ground beef and 55 mg of hair with no follicles was used. 950 uL digestion solution and 20 uL beta-mercaptoethanol (β-ΜΕ) was added. Heated at 990°C for 10 minutes. The fatty part of the beef dissolved. The muscle portion did not dissolve with heating. Added 50 uL Savinase. Within 30 minutes, the hair went from a gelatin- like consistency to a thin slurry in a matter of minutes. Digested at 56°C for 90 minutes. At this time, added 60 uL of kaolin particles followed by vortexing. Added 1 mL 100% EtOH. Centrifuged at 8000g for 2 minutes at 15°C. Added 1.4 mL 25 mg/mL Amresco pepsin to each. Started 37 °C digestion. After 1.25 hours digesting at 37 °C, both solutions were at pH 4. Rinsed once with 1.4 mL 70% 2PrOH. Rinsed twice with 1.4 mL deionized water. Rinsed twice with 1.4 mL of 10 mM Tris pH 8.0. Eluted for about 10 minutes into 1 mL 10 mM K₂HP0₄, pH 7.8. Pellets were eluted a second time with 500 uL of 40 mM Na₂HP0₄, pH 12. The melatin from the hair dissolved under these conditions. Figure 8 shows the UV spectra for the hair and ground beef. In the ground beef sample, the fat seemed to form a colloid, and, therefore, absorbance could not be measured. Table Table 6 shows UV absorbance numbers for samples in Example 7 and Example 8.

Table 6

Example 9 Nucleic acid isolation from pigeon fecal material

Small pieces of dried pigeon feces were placed in 1 mL digestion solution (GnHCl) and 20 uL beta-mercaptoethanol (β-ΜΕ). Used 50 uL Savinase. Digested overnight at 56°C. Dried pigeon feces contains large amounts of protein, approximately 26% by weight (according to The Use of Dried Pigeon Droppings in Rabbit Nutrition, by Nokhtar, S. M., et al.) Transferred contents of overnight digest to 2 mL polypropylene tubes. Added 60 uL kaolin particles. Vortexed. Precipitated with 1 mL 100% isopropanol (2-propanol, or 2PrOH). Digested for 1 hour at 37°C with 2 mL each tube 15 mg/mL pepsin in 5 mM HC1. The samples were eluted into 1 mL lOmM K₂HP0₄. Samples were diluted lOOx with the same in order to measure the absorbance.

Absorption spectra are shown in Figure 9. Note the very high absorbance at 290 nm, not 280 nm, as would be the case for aromatic amino acids in proteins. Also note the absorbance peak at 236 nm. There is also an absorbance minimum at 260 nm, but this means very little considering how much the sample had to be diluted to get an absorbance. Furthermore, uric acid has absorption peaks at 235 and 293nm. Example 10 Preparation of borate passivated ceria particles

Ceria was obtained from Inframat Advanced Materials Ce0₂ (58N-0802

IAM11139CEON). Ceria (123mg) was activated for passivation with borate by rinsing two times with 6 mL IN H₂SO₄. Centrifuged for 2 minutes at 200 g. Rinsed 3 times with 10 mL of 100 mM Na₂B₄0₇. Particles were stored in 10 mM Na₂B₄0₇.

Example 11 Preparation of phosphate passivated ceria and zirconia particles

For phosphate passivation of ceria and zirconia a "two-in-one" protocol was used. Ceria was from Inframat Advanced Materials. Zirconia was from Aldrich 544760-25g CAS 1314-23-4. Passivation was performed in 5 mL 1M NaH₂P0₄ dissolved in IN HC1. Zirconia and ceria were added such that the final concentration was 163 mM. Particles were incubated for 2 hours at room temperature with vortexing. The pH of the passivation solutions was pHl. After 2 hours, the particles were centrifuged at 2000g for 5 minutes. Particles were rinsed twice in 10 mL 1 mM NaH₂P0₄, pH 4. Example 12 Preparation of borate passivated zirconia particles

To a 25 g bottle of Aldrich Zr0₂ (544760-25g) add 100 mL INsulfuric acid. Split between two GSA centrifuge bottles. Centrifuge in GSA rotor in Sorvall centrifuge at 6000 RMP for 10 minutes. Discard supernatant. Add 200 mL 10 mM Na₂B₄0₇. Paint shake for 15 minutes. Centrifuge in GSA rotor in Sorvall centrifuge at 6000 RMP for 10 minutes. Discard supernatant. Repeat the steps of adding 200 mL 10 mM Na₂B₄C>7 and paint shaking for 15 minutes. Centrifuge in GSA rotor in Sorvall centrifuge at 6000 RMP for 10 minutes. Discard supernatant.

Example 13: Pepsin only digest of pooled normal human plasma

In this example pooled normal human plasma is digested with pepsin only. The following parameters were tested: 4 M urea with no β-ΜΕ; 6 M urea with no β-ΜΕ; 4 M urea with 1% v/v β-ΜΕ; and 6 M urea with 1% v/v β-ΜΕ.

This example was carried out as follows. Thawed about 5 mL of Bioreclammation BRH415419 pooled normal human plasma that had been aliquoted from a 50 mL frozen tube previously. In 2 mL microfuge tubes added 720 mg urea in tubes B and D and 480 mg urea in tubes A and C. 1300 uL thawed plasma into each tube.

Adjusted to pH 4-5 with 15 uL aliquots IN HC1. Took about 105 uL. Added 119 mg Amresco ProPure pepsin M142-250g lot 0019B466. Dissolved in 800 uL deionized water. Started digests by adding 200 uL pepsin solution. Added 20 uL β-ΜΕ to C and D after about 5 minutes. Added 2 mL with deionized water to A and C after about 10 minutes. Aliquoted 20 uL borate passivated AI2O₃ into 16 600 uL polypropylene microfuge tubes. Took 500 uL aliquotes from A-D at 30 minute intervals and vortexed with the AI2O₃ particles. This amounts to 325 uL original plasma. Checked pH with pHydrion 3-5.5 pH paper. All sampels were at about pH 4.5 to pH 5. Let bind for 30 minutes at room temperature. Microfuged for 20 seconds. Discarded the supernatant. Rinsed with 500 mL 5 mM glycine pH 3.7. Pipetted up and down twice with 100 uL pipetter. Microfuged for 20 seconds. Discarded supernatant. Rinsed with 500 mL 70% 2PrOH. Pipetted up and down twice with 100 mL pipetter. Microfuged for 20 seconds. Discarded supernantant. Pulse centrifuged for 30 seconds. Evaporated to dryness at 56°C. Harvested more at the 60 minute time point, allowed 30 minute for binding at room temperature, and rinsed as per the 30 minute time point. Also evaporated to dryness. Harvested and rinsed as per the other samples. After dried, eluted all samples in 108 uL 10 mM Na₂B₄0₇ pH 9. Eluted at 56°C for 30 minutes. Centrifuged for 1 minute.

Performed GAPDH PCR using 5 uL sample and a total reaction volume of 20 uL. Zeroed the Fisher GenesyslO spectrophotometer with 81 uL lOmM Tris pH 8 and 9 uL 10 mM Na2B407 (lOx dilution). Diluted 9 uL of the samples with 81 uL 10 mM Tris pH 8. Measured absorbance. Took 30 mL of the lOx dilution of the samples used for the absorbance measurements and diluted 1 :1 with PicoGreen and RiboGreen working stocks as per the manufacturer's protocol (Molecular Probes, Eugene, OR). Loaded 7uL sample and 7 uL PCR products in Lonza 2.2% gel. Used 2.5 uL Quantit standards, also from Lonza.

UV absorbance spectra are shown in Figure 10. The following observations were made. 1) There is a large absorbance in the 255-265 nm region. 2) The 260/280 nm ratios in Table 7 would be higher if the peak absorbance at 255 nm or 265 nm had been used instead of 260 nm. 3) The 260/230 nm ratio seems to approach it's optimal at 60 minutes of a room temperature digestion in pepsin. 4) The approximate concentration of RNA in the original plasma (accounting for the 3x concentration) is not as high as was measured in other examples in which there was an alcohol precipitation following a Savinase digestion. Figure 11 shows UV parameters plotted as a function of digestion time.

Concentrations of DNA and RNA were measured with Pico Green and Ribo Green.

Graphical representation of concentrations of both as a function of pepsin digestion time are given in Figure 12 and Table 7. Note that the absorbance around 260 nm

overestimates the amount of nucleic acids in the sample assuming that everything that absorbs at 260 nm is RNA. The fact that the absorbance peak is often slightly off suggests that there may be contamination with small RNA that may not bind RiboGreen with as high affinity as even the miRNA standard or that there may be purine nucleotides in the plasma, such as adenosine diphosphate from lysed red blood cells. Whatever this material is, eliminating the alchohol precipitation after the Savinase digestion and eliminating the Savinase digestion entirely seems to lessen this phenomenon.

A gel of input DNA is shown in Figure 13, and a gel of PCR products is shown in

Figure 14. Loading of lanes in Figure 13 and Figure 14 is as follows. Lane 0 - 2.5 uL Lonza quantit standards. Lanes 1-4 are from the 30 minute digestion with lane 1 - 4 M urea; lane 2 - 6 M urea; lane 3 - 4 M urea + 1 % β-ΜΕ; and lane 4 - 6 M urea + 1 % β- ME. Lanes 5-8 are from the 60 minute digestion with lane 5 - 4 M urea: lane 6 - 6 M urea; lane 7 - 4 M urea + 1% β-ΜΕ; and lane 8 - 6 M urea + 1% β-ΜΕ. Lanes 9-12 are from the 120 minute digestion with lane 9 - 4 M urea; lane 10 - 6 M urea; lane 11 - 4 M urea + 1% β-ΜΕ; and lane 12 - 6 M urea + 1% β-ΜΕ. Lanes 13-16 are from the overnight digestion with lane 13 - 4 M urea; lane 14 - 6 M urea; lane 15 - 4 M urea + 1% β-ΜΕ; and lane 16 - 6 M urea + 1% β-ΜΕ. The gel in Figure 14 also contains lane 17 as the negative control and lane 18 as the genomic DNA positive control.

In Figure 13, an arrow mark the material that migrates faster than the 1500 base pair Lonza standard and the 10,000 base pair Sigma broad range standard that is often seen in other examples and another arrow marks the material that migrates faster than the 100 base pair Lonza standard and the Sigma 50 base pair standard. Densities of the greater than 1500 base pair bands are given in Table 7.

Table 7 Summary of Results for Example 13

Digest condition A, 4M urea B, 6M urea C,4M urea + β-ΜΕ |Ρ, 6M urea + β-ΜΕ| Digest time 30 60 120 o.n. 30 60 120 o.n. 30 60 120 o.n. 30 60 120 o.n.

DNA in plasma, ng/mL 337 511 506 498 435 454 423 585 496 765 612 567 579 520 562 588

RNA in plasma, Ug/mL 4.5 4.8 4.7 4.4 4.7 5.3 4.5 4.7 7.0 4.7 4.6 4.7 6.7 5.1 4.9 5.5

260nm RNA, Ug/mL 5 8 10 11 10 8 9 13 5 12 14 9 10 7 9 3

260/230 0.61 0.63 0.62 0.53 0.56 0.6 0.64 0.58 0.69 0.64 0.63 0.61 0.67 0.68 0.7 0.55

260/280 1.4 1.3 1.4 1.4 1.4 1.3 1.3 1.4 1.3 1.4 1.4 1.4 1.3 1.4 1.4 1.4

>1500 base pairs band density 194 202 133 109 117 162 134 104 125 100 140 70 169 72 157 78

While adding any metal oxide nanoparticles to plasma tended to cause what appeared to cause clotting in spite of the anti-coagulants, there was no evidence of time dependent decrease in the greater than 1500 base pair band suggesting that depruination and strand breakage was not accelerated under these conditions. Addition of β-ΜΕ did not seem to enchance any parameter tested but at the same time did not seem to inhibit pepsin such that clotting resulted when the alumina was added.

In other examples, the Savinase digestion was reintroduced in order to reduce the protein load and to get the 260/280nm ratio closer to the expected value for RNA and/or DNA. The 70% 2PrOH rinse was changed to either an ethanol saline rinse (75% v/v) or 2PrOH (50% v/v) with 150 mM NaCl and 10 mM Tris pH 8 in order to obtain more consistent PCR product. Different metal oxides were explored in order to selectively isolate the larger DNA. Example 14

In this example, nucleic acids were captured with borate-passivated ceramic nanoparticles without an alcohol precipitation step after the Savinase digestion as was used in earlier examples. After the Savinase digest, the sample was adjusted to pH 4, digested with pepsin, and nucleic acids were captured with different varieties of borate- passivated ceramic nanoparticles.

This example was carried out as follows. 1.44 g urea was added to a 5 mL tube. 28 uL beta-mercaptoethanol was added for 100 mM in a volume of 4 mL. Newborn calf serum was thawed and added to the 4 mL mark as soon as it was thawed. 100 uL

Savinase was added for a one hour digestion at 56 C and serum pH, about pH 7.4.

400 uL of IN HCl was added to get pH 4, and 50 mg porcine pepsin was added for a final volume of 5 mL, at 4.8 M urea. The digest was carried out at room temperature for one hour after which it was frozen at -4°C.

Borate passivation was performed as described previously on Nd₂C>3, Ce0₂, Zr0₂, and AI2O₃ nanoparticles. Ce0₂, Nd₂C>3, and AI2O₃ nanoparticles were prepared according to Example 10, and Zr0₂ nanoparticles were prepared according to Example 12. The volumes of nanoparticles added to the pepsin digests were normalized to the OD₄oo_nm- The newborn calf serum was thawed and 120 uL was aliquoted into 600 uL microfuge tubes. Particles were added such that 10 uL

per each of four replicates for the four types of particles. The mixture was briefly vortexed and incubated at room temperature for 30 minutes. At the end of 30 minutes, 450 uL of 5 mM glycine, pH 3.3, was added to each tube with brief vortexing. Samples were centrifuged in a Fisher 235 C table top centrifuge for 15 seconds. Pellets we re-rinsed once with 0.5 mL of 5 mM glycine, pH 3.3, and rinsed once with 0.5 mL of 70% 2-propanol. Each change of solution was followed by gentle pipetting and then centrifugation in the Fisher centrifuge for 15 seconds. Residual 70% 2-propanol was allowed to evaporate to dryness from the uncapped tubes at 52°C. Samples were eluted into 0.001% Tween-20 10 mM Na₂B₄0₇, pH 9.

For analysis, the spectrophotometer was zeroed with a lOx dilution of the elution buffer in 10 mM Tris, pH 8. Samples were eluted at 52°C for the indicated times. Brief vortexing was carried out every 5 minutes. The 30 minute elution set of samples was centrifuged for 15 seconds in the Fisher table top centrifuge as the others were eluting. Samples were diluted lOx with 10 mM Tris, pH 8, to measure the absorbance, i.e. 9 uL sample to 81 uL Tris. After the UV absorbance was measured, 30 uL of this diluent was taken for PicoGreen (DNA) and RiboGreen (RNA) (Invitrogen, Molecular Probes, Eugene, OR) measurements. These 30 uL aliquots were mixed 1:1 with PicoGreen and RiboGreen working stocks. An overnight GAPDH PCR reaction with 5 uL of sample to a reaction volume of 20 uL was performed.

Table 8 UV parameters

Table 8 presents basic UV parameters. The concentration of RNA was estimated assuming an extinction coefficient of 0.025 ug^mLcm^"1. Adjustment was made for the 10x dilution. Note that elution times longer than 30 minutes are not required.

DNA and RNA concentrations as measured by PicoGreen and RiboGreen are shown in

Table 9. The dilution factor has been accounted for.

The PCR input DNA (and RNA) is shown in Figure 15. Note that only the A1₂0₃ isolated samples are readily visible on a Lonza 2.2% agarose gel that is selective for DNA but will also bind to RNA with lower affinity. The density of staining in lanes shown in Figure 15 was quantified with Scion Image. Density, in arbitrary units, is shown in Table 10. Qualitatively, the gel densities correspond with PicoGreen measurements of DNA concentration. Over twice as much DNA and RNA from AI2O₃ and Zr0₂ was seen than from Ce0₂ and Nd₂C>3. Figure 16 shows results for the results from GAPDH PCR with an expected 451 base pair product.

Table 9 RNA and DNA concentrations measured by RiboGreen and PicoGreen

Table 10 Scion Image band density values of input DNA

For Figures 15 and 16, the gels lanes are loaded as follows: 0 - Sigma broad range ladders; 1 - AI2O₃ 30 minute elute; 2 - AI2O₃ 60 minute elute; 3 - AI2O₃ 90 minute elute;

4 - AI2O₃ 120 minute elute; 5 - Ce0₂ 30 minute elute; 6 - Ce0₂ 60 minute elute; 7 - Ce0₂ 90 minute elute; 8 - Ce0₂ 120 minute elute; 9 - Nd₂0₃ 30 minute elute; 10 - Nd₂0₃

60 minute elute; 11 - Nd₂0₃ 90 minute elute; 12 - Nd₂0₃ 120 minute elute; 13 - Zr0₂ 30 minute elute; 14 - ZrC>2 60 minute elute; 15 - ZrC>2 60 minute elute; 16 - ZrC>2 120 minute elute; 17 - negative control; 18 - gDNA positive control.

As seen in Figure 16, except for lane 4, the 120 minute elution from AI2O₃, none of these samples yielded a visible 451 base pair GAPDH PCR product. Likewise, none of the samples isolated with ZrC>2 yielded and PCR product, see lanes 13-16 of Figure 16.

All of the samples isolated from CeC>2 and Nd2C>3 yielded PCR product to some extent. The yield of nucleic acids using AI₂O₃ particles was greater than the yield seen with the other particles tested. In subsequent examples, it appears that the < 50 base pair material may be a source of PCR inhibition. Elution times of more than 30 minutes did not result in a greater yield or a cleaner product.

Example 15

In this example, a comparison was made among various particle types including AI₂O₃, NiO, T1O₂, and WO₃, see Table 11. The purpose was to compare particles of metal oxides with a broad range of isoelectric points (pi or IEP). Tungsten oxide (WO₃) has an IEP in the low range; alumina (AI₂O₃) and titania (T1O₂) have IEPs in the midrange; and nickel oxide (NiO) has an IEP in the high range. Nucleic acid yield by the low IEP particle, WO₃, was minimal. The nucleic acid isolated with the NiO and T1O₂ particles yielded the best PCR products. Of the three particles that bound DNA/RNA well, only NiO did not appear capture the less than 50 base pair material.

Table 11

This example was carried out as follows. Starting material was a dual protease digest of BioReclammation pooled normal human plasma. 150 uL was aliquoted among sixteen 600 uL polypropylene tubes. The volume of particles added was normalized to the optical density at 400nm (OD4oonm) with the starting point of 10 uL of alumina,

OD4oonm = 50 in replicates of four and 16 tubes total. Samples were incubated at room temperature for 30 minutes. Samples were centrifuged in a Fisher 235 C table top centrifuge for 20 seconds. Supernatant was discarded. A 200 uL pipette and tip were used to remove what adhered to the sides of the tube when the tube was inverted and given a gently shake. Samples were rinsed once with 500 uL of 5 mM glycine, pH 3.3, and pipetted up and down four times with pipette set on 150 uL. Samples were centrifuged in Fisher table top centrifuge for 20 seconds. Supernatant was discarded. Using 15 mL EtOH saline, 150 uL 1M Tris, pH 8, was added for 10 mM Tris. All pellets were rinsed with 500 uL, and the supernatant was discarded. Samples were allowed to dry at 52°C in uncapped tubes for 20 minutes. Samples were eluted with 100 uL of 10 mM Na2B₄07 with 0.001% Tween 20. Note the volume decrease from the original 150 uL in 6M urea.

The spectrophotometer was zeroed with 81 uL 10 mM Tris, pH8, and 9 uL of the elution buffer. 9 uL of sample was diluted with 81 uL of 10 mM Tris, pH 8, and the absorption was measured. Only the samples isolated from the WO₃ nanoparticles had to be centrifuged a second time to measure the UV absorbance. Even after 1-5 minutes of centrifuging in the Fisher centrifuge many of the WO₃ samples were still turbid. One possibility may be that lipid was carrying over.

30 uL of sample was mixed 1:1 with PicoGreen and RiboGreen working stocks as per the protocol. Each successive elution was about 20 to 25 minutes longer than the previous. A 2.2% agarose gel from Lonza was used to resolve raw DNA (Figure 17) and PCR products (Figure 18) Figure 17 and Figure 18 contain lane 0 - Sigma wide range standards; lanes 1-4 - AI2O₃, 30-95 minute elutions; lanes 5-8 - NiO, 30-95 minute elutions; lanes 9-12 - T1O2, 30-95 minute elutions; and lanes 13-16 - WO₃, 30-95 minute elutions. When most of the PCR products had run down the gel, the negative and positive controls were loaded in the upper right hand corner of the gel. There were no PCR products in the WO₃ samples. The gel plot 2 algorithm of Scion Image was used to measure band density.

UV absorbance values are shown in Table 12 and provide an index of sample quality. No correlation in elution time and UV parameters was noted in any of these samples (not shown). The average 215 nm and 350 nm signals are presented as an index of possible particle carryover. On this basis, tungsten oxide was determined to be a less preferred particle type. Tungsten oxide also had the lowest IEP of the particles tested in this example (see Table 11). Table 12 UV absorbance values

The NiO samples seem to have less absorbance at 260 nm (not shown but reflected in the approximate RNA concentration, Table 12). Both the AI2O₃ and the NiO particles yield samples with fairly good 260/280 ratios suggesting that protein contamination is minimal.

Table 13 RNA and DNA concentrations from PicoGreen and RiboGreen

The PicoGreen and RiboGreen results are shown in Table 3. The AI2O₃ and T1O2 nanoparticles recovered more RNA than the NiO nanoparticles as shown in Table 13. The total RNA isolated with AI2O₃ nanoparticles does not appear to be significantly greater than that isolated with NiO nanoparticles (see Table 12), though there is more of the material that migrates faster than the 100 base pair standard, Figure 18 and Table 13.

Figure 17 is a gel of PCR input DNA (and RNA). The lanes in Figure 17 are loaded as follows: lane 0 - Sigma wide range standards; lanes 1-4 - AI2O₃, 30 to 95 minute elutions; lanes 5-8 - NiO, lanes 30 to 95 minute elutions; lanes 9-12 - T1O2, 30 to 95 minute elutions; lanes 13-16 - WO₃, 30 to 95 minute elutions. The arrows in

Figure 17 mark the degraded DNA and RNA that migrate faster than 100 base pah- standard and the fragment that migrates a little bit slower than the 1500 base pair standard. There appears to be genomic sized DNA in at least three of the WO₃ isolated samples, see Figure 17.

Figure 18 shows the results after performing PCR directed to GAPDH, a 451 base pair product, on the samples isolated in this example. The lanes in Figure 18 are loaded as follows: lane 0 - Sigma wide range standards; lanes 1-4 - AI2O₃, 30 to 95 minute elutions; lanes 5-8 - NiO, lanes 30 to 95 minute elutions; lanes 9-12 - Ti0₂, 30 to 95 minute elutions; lanes 13-16 - WO₃, 30 to 95 minute elutions; lane 17 - H₂0 negative control; lane 18 - genomic DNA positive control. In Figure 18|note the relatively clear PCR products for NiO-isolated samples (4 of 4 replicates, lanes 5-8) and for Ti02 (2 of 4 replicates, lanes 10 and 12). The bottom gel image taken later during the electrophoresis process was used to quantify the PCR product.

Gel densities determined by Scion Image are shown in Table 13. Only alumina and titania yielded any material that migrated faster than the 50 base pair standard. These data counter the observation in other examples that the small material may correlate with PCR inhibition. Variable PCR products were seen with titania in spite of the large amounts of material that migrated faster than the 50 base pair standard. None of this material was seen with the WO₃ isolated samples. One possibility is that the WO₃ nanoparticles damage the DNA in some way that prevents good amplification of the GAPDH product.

As seen in this example, NiO nanoparticles yielded DNA that performed well in PCR with very little < 50 base pair material; and Titania yielded DNA that performed well in PCR and a significant amount of < 50 base pair material.

Table 13 Scion Image Gel band densities

Example 16

This example examines the utility of metal oxides that are soluble under the 1 N sulfuric acid conditions of borate passivation, for example La₂0₃ and MgO. This example illustrates that La₂0₃, MgO, and NiO particles passivated with glycine at pH 4 are each able to yield isolate DNA from plasma that performs well in PCR. The yield of isolated DNA greater than 1500 base pairs was similar to the yield of DNA less that about 100 base pairs. Table 15 pKas of glycine and other acids

Advantages of using particles with high IEP have been observed. Some nanoparticles with high IEP like, La₂0₃ and MgO, however, dissolve under the standard conditions used to passivate with borate, IN sulfuric acid. Glycine at pH 3.3 to pH 4 is a desired rinse solution and buffer, and it is also inexpensive. In this example, its utility as a passivating agent is explored. This example further explores the utility of altering the type of nucleic acid isolated from the dual protease digest by altering surface

characteristics of the metal oxide.

This example was carried out as follows. 100 mg of Inframat 57N-0801 lot IAM7158NLAO lanthanum(III) oxide La₂0₃, IEP = pH 10, was placed in a 2 mL polypropylene tube. 1 mL 5 mM glycine, pH 3.3, and 100 uL 1 M sulfuric acid was added to reach a pH of about 4. La₂C>3 did not dissolve appreciably. This protocol was repeated for magnesium oxide (magnesia) MgO (IEP= 9.8-12.7, see Chemical Properties of Material Surfaces, Marcel Dekker, 2001.) and NiO. MgO was never successfully titrated to pH 3.3. Particles were rinsed twice with 5 mM glycine, pH 3.3. 150 uL of a dual protease digest of plasma that had been stored frozen at -4°C in 16 600 uL polypropylene tubes was aliquoted. In replicates of four, each tube got 10 uL of borate- passivated NiO and 10 uL of the glycine (and sulfate) passivated nanoparticles. Note that the borate passivated nanoparticles are technically also sulfate passivated because sulfuric acid was used to adjust the pH. Samples were incubated at room temperature for 15 minutes. Samples were centrifuged in a Fisher 235 C centrifuge for 15 seconds. Samples were rinsed with 500 uL 5 mM glycine, pH 3.3. Samples were rinsed once with 10 mM Tris pH 8 EtOH saline rinse. EtOH in pellets was allowed to dry in uncapped tubes to evaporate to dryness at 52-56°C. Samples were eluted with 100 uL of 10 mM Na₂B₄07 with 0.001 % Tween 20. Note the volume decrease from the original 150 uL in 6M urea. The spectrophotometer was zeroed with 81 uL 10 mM Tris pH8 and 9 uL of the elution buffer. 9 uL of the sample was diluted with 81 uL of 10 mM Tris pH 8 and measured the absorption. Took 30 uL of sample was mixed 1 : 1 with each of PicoGreen and RiboGreen working stocks as per the protocol. Each successive elution was about 20-25 minutes longer than the previous. A 2.2% agarose gel from Lonza was used to resolve input DNA (middle tier) and PCR products (top tier). Sigma ladders were used as the standard in the gel for the PCR products, and 2.5 uL of the Lonza Quantit standards were used for the input DNA gel. When most of the PCR products had run down the gel, the gel was loaded with the negative and positive controls in the upper right hand corner of the gel. The gel plot 2 algorithm of Scion Image was used to measure band density. UV absorbance was measured. In this example all comparisons were in reference to the glycine-passivated NiO nanoparticles. All of these spectra have high absorbance at 350 nm suggesting light scattering of particles. The estimated RNA concentrations are substantially different from those measured by RiboGreen as shown in Table 17. Note that NiO-glycine is the benchmark for two tailed paired t-tests in these comparisons. The 260/280 ratios seem high in samples isolated from both types of NiO particles, see Table 16. Some of the La₂0₃ might be dissolving. The apparent poor quality of the spectra from the La₂0₃-Gly and MgO-Gly isolated samples are bad enough to give reason to not use glycine passivated forms of these particles. On the other hand, not everything that absorbs at 260 nm in blood plasma is nucleic acid. Adenosine diphosphate released from red blood cells might also bind to and elute from metal oxide nanoparticles.

Table 16 UV parameters

Table 17 PicoGreen and RiboGreen estimations of DNA and RNA Concentrations

It is interesting to note that even though the UV spectra of samples isolated from La₂0^"3-Gly and MgO-Gly indicate a lower purity of nucleic acids, the concentrations of DNA and RNA are about the same, Table 17. The most likely explanation is that La₂0^"3- Gly and MgO-Gly are dissolving or have broken into smaller fragments.

The isolated DNA input into the PCR reactions (sometimes referred to as "input DNA") is shown in Figure 19. Two images were taken during the electrophoresis to demonstrate that La₂0^"3-Gly seemed to isolate the most material less than about 100 base pairs even though gel quantitation demonstrated otherwise, see Table 18. Also note that the background was darker for the La₂0^"3-Gly samples. Also shown is that the greater than about 1500 base pair band was statistically of about the same density for all sixteen samples, again with a rather large standard deviation, Table 18.

Table 18 Scion Image Band Density

Figure 19 shows the DNA isolated in this example. The lanes in the gel in Figure 19 are loaded as follows: lane 0 - 2.5 uL Lonza Quantit standards; lanes 1-4 - N1O-B₃O7 isolated samples; lanes 5-8 - NiO-Gly isolated samples; lanes 9-12 - La₂0^"3 isolated samples; lanes 13-16 - MgO-Gly isolated samples. The top panel is an image of the gel early in the electrophoresis process. The bottom panel is an image of the same gel later in the electrophoresis process.

In Figure 20, there are two images that were taken of the sample gel containing the GAPDH 451 base pair amplicons from the PCR reactions done on the samples.

Figure 20 shows PCR products from the samples isolated in this example. The lanes were loaded as follows: lane 0 - Sigma broad range standards; lanes 1-4 - N1O-B₃O7 isolated samples; lanes 5-8 - NiO-Gly isolated samples; lanes 9-12 - La₂0₃ isolated samples; lanes 13-16 MgO-Gly isolated samples; right panel lane 17 - deionized water negative control; right panel lane 18 - genomic DNA positive control. The top panel is an image of the gel early in the electrophoresis process; the bottom panel is an image of the same gel later on in the electrophoresis process. Some aberrations were seen around 200 base pairs that were resolved as the bands continued to move along the gel. These aberrations around 200 to 300 base pairs are frequently seen with samples isolated using multiple particle types. They are not seen with the positive and negative controls.

In this example, all four formulations of nanoparticles tested yielded DNA that worked well in PCR reactions and was able to yield the GAPDH PCR product. Glycine pretreatment, or passivation, of two types of acid soluble nanoparticles (La₂0₃ and MgO) resulted in a product that was able to isolate DNA and RNA from blood plasma. While UV spectra might indicate issues with these nanoparticles, use of these glycine treated metal oxides might have other utilities in non-nanoparticle formulations. La₂C>₃ has optical and semi-conductor uses that might be combined with its ability to bind nucleic acids. Differences in the amount of DNA and RNA isolated and the size of the nucleic acids isolated, based on the particle type used, were also seen in this example.

Example 17

This example shows a comparison between borate passivated kaolin

(Al₂Si₂0₅(OH)₄) and borate passivated talc (Mg₃Si₄Oi₀(OH )₂). Both of these natural minerals were used to isolate nucleic acids in a dual protease digest indicating that other natural minerals are likely to be of utility as well.

In other examples (Example 16, Example 21), MgO was shown to isolate genomic sized DNA while sparing nucleic acids that migrate faster than the 50 base pair DNA standard in a 2.2% agarose gel. One issue with the use of MgO is its solubility in mild acid. Talc is the magnesium-based homo log of kaolin which is aluminum-based. Both have a sheet like structure with alternating layers of silica and aluminum and magnesium oxides. Both talc and kaolin have the advantage of economy and a sheet-like structure that packs easily into pellets. In this example, baby powder from a local drug store was used as the source of talc. The only ingredients were talc and fragrance. A Na2B₄07 (borax) passivation protocol was used for both nano-sized kaolin as well as "baby powder" talc of an unknown particle size. Market variety kaolin (fluoride and phosphate passivated) and micron sized borax passivated kaolin were added to the comparison.

This example was carried out as follows. 0.8g of talc was placed in a 15 mL polypropylene tube. 0.8g of kaolin, Englehard, ASP ultra fine lot 02024 was placed in a separate 15 mL polypropylene tube. 1M H₂SO₄ was added to these tubes for a total volume of 12 mL. Tubes were vortexed and centrifuged at 2000xg for two minutes. Deionized water was added for a total volume of 12 mL with vortexing. The talc resuspended very well. The kaolin clumped. The tubes were centrifuged at 2000xg for 2 minutes, and the particles were rinsed three times with 12 mL of 100 mM Na2B₄C>7. Particles were resuspended by adding 1 mL of 100 mM Na2B₄C>7 and water for a final volume of 5 mL. A volume of 6 uL of the borate passivated talc was dispensed into each of four 600 uL polypropylene microfuge tubes. A volume of 3 uL of nano-kaolin-

Na2B₄C>7 was dispensed into four 600 uL tubes, and a volume of 3 uL of micro-kaolin- Na2B₄C>7 was dispensed into four 600 uL tubes. Used 10 uL market grade kaolin and 100 uL of a pooled normal human plasma dual protease digest.

The protocol for pooled normal human plasma dual protease digest is as follows. Thawed a tube of pooled human plasma from BioReclammation. Used 1.8 g urea for 5 mL. For 6 M urea, used 35 uL β-ΜΕ for 100 mM. Seven tubes total. Used 200 uL of Savinase for the digest. For seven tubes, added plasma as it melted (almost to a slushy slurry of partially frozen) to the 1.8 g urea to almost the 5 mL mark. Then added the β- ME and Savinase. Started the digest, and digested on a heat block at 56°C. Transferred samples to the freezer after 90 minutes.

Thawed a tube of plasma that had been digested with Savinase. Took 600 uL 1 M H₂SO₄ to get the 5 mL of Savinase only digest to pH 4.5 as judged by pH Hydrion 3-5.5 pH paper. Added 65 mg of Amresco porcine stomach pepsin. Digested at room temperature for 1 hour. After one hour, put in freezer for overnight storage.

The protocol for micro-sized kaolin is as follows, lg kaolin GA-2 (Source Clay

Repository) in 50 mL tube with 1 M H₂SO₄ to the 50 ml mark. Rinsed once with 50 ml 1 N H₂SO₄. Used a plastic transfer pipette to break up the aggregates. First borate rinse is to bring volume to 20 ml with 100 mM borate. Deionized water to 50 mL. Second borate rinse is the same as the first. Third borate rinse used 100 mM borate to 10 ml mark then to 50 ml mark with deionized water.

Samples were vortexed and allowed to sit at room temperature for 15 minutes. Samples were centrifuged in a Fisher 235 C centrifuge for 20 seconds, and the supernatants were discarded. Samples were rinsed twice with 500 uL 5 mM glycine, pH 3.3. Samples were then rinsed once with 500 uL 10 mM Tris pH 8 in 75% v/v EtOH and 150 mM NaCl. Samples were allowed to dry for 30 minutes. Samples were eluted into 50 uL 10 mM Na₂B₄07 and 0.001% Tween 20 and centrifuged for 2 minutes or longer. A volume of 9 uL of the samples was diluted with 81 uL 10 mM Tris pH 8 to measure the UV absorbance. Two samples of 30 uL of this material were diluted, one with 30 uL PicoGreen working stock and one with 30 uL RiboGreen working stock. A volume of 9 uL sample with 1 uL loading dye was evaporated to about 3 uL. Samples were loaded in the gel by grouping the replicates together.

UV absorbance is shown in Table 19. The 260/230 ratios are substantially below 1.0 as seen in Table 19. All of the 260/280 ratios are below the optimal value of 1.8. The 260 nm absorbance estimated concentration of RNA in the isolated nano-kaolin-B₄C>7 samples does not correlate well with the RiboGreen values should in Table 20. The values in Table 19 have been adjusted for the tenfold dilution. Other 260 nm estimates of the RNA concentration correlate well with the values obtained with RiboGreen.

Table 19 UV Absorbance

Table 20 PicoGreen and RiboGreen measurements of DNA and RNA

It is also interesting that that was not a significant difference in the yields of DNA or RNA from any of the particles. Contrast this to the gel, Figure 21, showing what appears to be less material that migrates faster than the 50 base pair DNA standard. Gel denistometry with Scion Image supports this appearance. The amount of approximately 10,000 base pair DNA is not substantially different between the four groups.

Figure 21 is a gel of DNA isolated from lanes A - talc-B₄07; lanes B - nano- kaolin-B₄C>7; lanes C - micro-kaolin-B₄C>7; and lanes D - market variety kaolin.

Table 21 Gel band densities from Scion Image

The results of this example suggest that borax passivated talc results in less recovery of small RNA (and DNA) Also, a metal oxide containing mineral in addition to kaolin has been demonstrated to be able isolate DNA and RNA from a dual protease digest. As more is known about individual metal oxide chemistries using pure nanoparticles, additional metal oxide containing minerals can be used for their specific applications and selectivity.

Example 18

In the BioReclammation pooled normal human plasma, the nucleic acid present is primarily greater than 1500 base pair bands and less than 50 to 100 base pair bands. In this example, the goal was to have a broad size distribution of degraded DNA to better understand what sizes of DNA fragments NiO-based particles are effective in isolating and what sizes of DNA fragments NiO-based particles tend to leave in the sample. There is not always a sufficient amount of DNA from plasma between 50 and 1500 base pairs to visualize on a gel. For this purpose, beef heart was obtained from a local grocer. Pooled normal human plasma from Bioreclammation was also used in this example.

Additionally, 10 mL of 100 mg/mL unpassivated NiO was suspended in mineral oil. Using mineral oil was a simple method of adding unpassivated NiO, and mineral oil is thought to coat the nanoparticles non-specifically until being displaced by chemical bonds between metal oxide groups on the particles and phosphate groups on the DNA/RNA. It is hypothesized that adenosine diphosphate and other possible phosphate containing compounds in plasma may be less likely to displace mineral oils than nucleic acids which have multiple phosphate groups to bind to the metal oxide groups on the nanoparticles.

This example was carried out as follows. 1.8 g of urea was placed in a 15 mL tube. 248 mg frozen beef heart was cut off the frozen beef heart with scissors. Water was added for a total volume of 3.5 mL. The sample was heated in a heat block to 56°C. Previously 100 mM β-ΜΕ had been used for the plasma dual protease digest. Since β- ME degrades faster with increasing temperature, 1% v/v beta- ME is used in this example. 1% v/v β-ΜΕ is generally used for denaturing proteins before they are resolved on polyacrylamide gels. 35 uL of β-ΜΕ was added when the temperature of the heat block reached 80°C. After twenty minutes, the pH of the digest was checked pH paper. 250 uL of 20x extraction buffer was added, and the pH measured pH 9 with pH paper. (Note that this is a variation of the dual protease digest procedure used with plasma because the beef heart was diluted to compensate for the higher per protein content.) The sample was digested for 1.5 hours at 56°C. Then about 80 mg pepsin was added and the volume was adjusted to 7.5 mL and the pH was adjusted to pH 4. Digest was run for an additional 1.5 hours at room temperature. The pH was between about pH 4 to pH 4.5. Samples were centrifuged for 2 minutes. There was a protein pellet present in the heart digest sample. There was no pellet present in the plasma digest sample, but there did appear to be a lipid layer present. Did not use this for the NiO extraction. 10 mL of the NiO in mineral oil, lOOmg/mL w/v was aliquoted into eight 600 uL microfuge tubes. A volume of 350 mL of the centrifuged heart digest was aliquoted into four 600 mL tubes and 350 uL of the plasma digest was aliquoted into another four 600 mL tubes. The NiO was mixed in by pipetting up and down five times with the pipetter set at 100 uL. The pH was checked for the last samples mixed. The heart digest was about pH 4.5, and the plasma digest was about pH 4. After 230 minutes at room temperature, the samples were centrifuged in a Fisher table top centrifuge for 2 minutes. Supernatants were transferred to 600 uL tubes with 4 uL of phosphate passivated. The NiO pellets were rinsed with 400 uL of 5 mM glycine, pH 3.7. Samples were mixed by pipetting up and down with a pipetter set at 160 uL. Samples were rinsed with 400 uL of 49% 2-propanol rinse with 150 mM saline and 10 mM Tris, pH 8. Rinsing was repeated for kaolin capture. Samples were evaporated to dryness at 56°C. Samples were eluted with 50 uL of 10 mM Na₂B₄07 at 56°C. After 5 minutes of rehydration, samples were pipetted up and down five times. Then, samples were vortexed periodically as detailed below.

A Thermo Scientific Genesys UV spectrophotometer was zeroed 85.5 uL of 10 mM Tris mixed with 4.5 uL 10 mM Na₂B₄07, the same buffer that was used to elute DNA from the NiO and kaolin nanop articles. Replicate sets were eluted for

approximately 30, 55, 80, and 105 minutes, i.e. replicates #1, #2, #3, and #4. Replicate sets were centrifuged for 2 minutes after the designated elution times. Diluted 4.5 uL into 84.5 uL 10 mM Tris for the UV readings. Vortexed other samples during this time. The samples used for used for UV readings were diluted 1:1 with PicoGreen and RiboGreen working stocks as per the Molecular Probes protocol (Eugene, Oregon).

Undiluted samples (5 uL) were subjected to PCR using GAPDH primers obtained from Integrated DNA Technologies. On parafilm, about 1 to 2 uL of Lonza gel loading buffer and 20 uL of the samples were pipetted in order to concentrate the samples for gel electrophoresis. 2.5 uL of Lonza Quantit standards, 6 uL of the sample PCR products, and all of the 20 uL of concentrated input DNA were loaded in a Lonza 2.2 % gel. Gel images at various stages of the electrophoresis process were imported into Scion Image. The gel plot 2 macro was used to define peaks and regions within peaks for comparing amounts of DNA or RNA resolved on gels, see Figure 23.

UV absorbance is shown in Table 22. PicoGreen and RiboGreen measurements of DNA and RNA concentrations are shown in Table 23. The PicoGreen and Ribo Green readings are more reliable than the UV absorbance of the 20x dilution of the samples. There is a surprising amount of RNA in the NiO extracted plasma samples that does not seem to be visible on the DNA gel shown in Figure 23. In kaolin recovery samples from both the heart and the plasma, there is about 30 fold more RNA than DNA. For NiO capture, the ratio is much greater.

Table 22 UV Absorbance

Table 23 PicoGreen and RiboGreen measurements of DNA and RNA concentrations

Input DNA and small RNA can be resolved by a 2.2% agarose Lonza gel and is shown in Figure 22. Notable, there is a broad size distribution in the nucleic acids isolated from bovine heart. Figure 22 is a gel of DNA isolated in this example (input DNA) resolved on a 2.2% agarose gel. The lanes of the gel were loaded as follows: lane 0 - 2.5 uL Lonza Quantit marker; lane 1 - NiO plasma #1 ; lane 2 - NiO heart; lane 3 - kaolin recovery plasma; lane 4 - kaolin recovery heart; lane 5 - NiO plasma #2; lane 6 - NiO heart; lane 7 - kaolin recovery plasma; lane 8 - kaolin recovery heart; lane 9 - NiO plasma #3; lane 10 - NiO heart; lane 11 - kaolin recovery plasma; lane 12 - kaolin recovery heart; lane 13 - NiO plasma #4; lane 14 - NiO heart; lane 15 - kaolin recovery plasma; lane 16 - kaolin recovery heart. Figure 22 A is the get early in the

electrophoresis process, emphasizing the lack of capture of less than 100 base pair material with NiO and the recovery of this material with kaolin. Figure 22B is the same gel later in the electrophoresis, indicating that NiO did not capture all larger DNA in the heart sample.

The bands in Figure 22A were imported into Scion Image for densitometry. Table 24 shows Scion Image values for gel band densities. In the first half of Table 24, showing densities for greater than 100 base pair material, there is a tendency for NiO in mineral oil to capture a only about one-half (plasma) to two-thirds (heart) of the greater than 100 base pair material. For the less than 100 base pair material, shown in the bottom half of Table 24, about the same amount is captured by NiO for both the heart and plasma. Note that there is more material less than 100 base pairs in the plasma and that it is possible to select the recovered nucleic acid according to size. Table 24 Scion Image values for gel band densities

Figure 23 shows PCR results using the DNA isolated in this example. The expected size of the GAPDH amplicon is 451 base pairs. The lanes of the gel are loaded the same as in Figure 3 with the addition of lane 17 as negative control and lane 18 containing human genomic DNA as a positive control. In Figure 23, there was some degree of PCR product for all samples except for the negative control in lane 17. In three (lanes 1, 9, and 13) of the four plasma samples isolated with NiO, there is very visible PCR product. Even in lane 5, it is slightly visible. In the kaolin recovery (done after the NiO recovery) from the plasma, lanes 3, 11, and 15, it is also faint.

Sizing of DNA and/or RNA can be achieved by use of NiO for the initial capture and kaolin recovery of what NiO missed. Further size discrimination may be achieved with a 70% 2-propanol of the kaolin rather than the 50% 2-propanol, 150mM NaCl, lOmM Tris pH 8 used in this example. In other examples, particles were rinsed with 70% 2PrOH. This resulted in less predictable PCR product and what appears to be proportionally more greater than 100 base pair material that is DNA and RNA.

Example 19: Utility of automation

This example shows that DNA useful for downstream applications can be isolated using mixed ferric oxides of MFe₂03 where M is Co, Mn, Ni, or Zn. This example shows that the dual protease digest can be used to extract DNA and RNA from pooled human plasma without the use of a centrifuge. Magnetic nanoparticles are extensively used as MRI contrast agents. Conventional iron oxide based contrast agents such as

superparamagnetic iron oxide (SPIO) and related nanoparticle probes (e.g., cross-linked iron oxide (CLIO)) have limited uses for advanced MR imaging due to their relatively poor magnetic contrast effects (i.e., low magnetic moment and also low r 2 coefficient). In recent years, magnetism-engineered iron oxides (MEIO) have been explored as contrast agents in magnetic resonance imaging (Jun 2008). CoFe₂0₄/DNA structures are also discussed in this review (Jun 2008). (See, Jun Y-W, Seo J-W, Cheo J (2008) Nanoscaling laws of magnetic nanoparticles and their applicabilities in biomedical sciences. Accounts of chemical research, 41(2) 179-189.)

The aim of work using many of the same mixed ferric oxides as used in this example was to investigate the electric double layer of magnetic fluids, or the layers of surface charges that could impact DNA/RNA binding, of CoFe₂0₄, MnFe₂0₄, ZnFe₂0₄, NiFe₂0₄, and CuFe₂0₄ of different sizes by means of the small angle X-ray scattering technique (Itri 2001). (See, Itri R, Depeyro J, . Tourinho F.A, . Sousa M.H (2001) Nanoparticle chain-like formation in electrical double-layeredmagnetic fluids evidenced by small-angle X-ray scattering. Eur. Phys. J. E 4, 201-208.)

Magnetic particles (MnFe₂0₄, ZnFe₂0₄ NiFe₂0₄, CoFe₂0₄) come from Inframat Advanced Materials. Tared out 100-200 mg and placed in 15 mL polypropylene tubes, one for each for each magnetic particle type by itself. Metal oxide groups were activated by soaking in 10 mL 1 N sulfuric acid. Particles were rinsed twice with 10 mL deionized water until the pH was between 3.5 to 3.7. Particles were soaked in 10 mL 1 M Na₂B₄C>7 for one hour and resuspended in a final volume of 100 mM Na₂B₄C>7 such that the concentration of particles was 50 mg/mL. In 600 uL polypropylene tubes, 20 uL of the particles and 150 mL of a dual protease digest of pooled normal human plasma were mixed by vortexing. Started separating the first set after 15 minutes at room temperature. Samples were rinsed with 1 mL 5 mM glycine, pH 3.7, and 1 mL of the 2-propanol saline rinse with 10 mM Tris, pH 8. Samples were evaporated to dryness. Samples were eluted into 100 uL of 10 mM Na₂B₄C>7, pH 9. Approximate elution times for replicate sets 1-4 were 30, 60, 90, and 120 minutes.

The UV absorbance was measured without dilution except for replicates 1 -3 of NiFe₂0₄, replicate 3 of MnFe₂0₄, and replicate 4 of CoFe₂0₄. These were diluted 10 fold. The absorbances were adjusted accordingly in Figure 24. A volume of 7 uL of sample was loaded in a 2.2% agarose Lonza gel using 2.5 uL of Lonza Quantit standards. Performed standard GAPDH reaction with 5 uL sample and total reaction volume of 20 uL. Gel band density was measured in Scion Image using the gelplot 2 macro.

Figure 24 shows UV absorbance spectra of the nucleic acids isolated using the magnetic beads described in this example. At all time points of elution (and binding), the NiFe₂04 particles exhibited the greatest signal in the short UV region around 220 nm, see Figure 1. The 260/230 nm ratio index of purity is not that different from the other particles tested, see Table 25. With the MnFe₂C>4 particles, the nucleic acid peak at 260 nm was better defined with lower binding times and elution times, see Figure 24. This did not, however, translate into better 360/230 and 260/280 ratios.

There was not much UV absorbing material captured and eluted from the ZnFe₂C>4 particles, see Figure 24. UV data from Figure 24 are shown in tabular form in Table 25 (per time point) and Table 26 (average of time points + standard deviation). In this example, the results indicate that longer binding/elution times did not result in higher quality spectra.

Figure 25 contains graphs of UV values. Figure 25 A shows the approximate RNA concentration assuming all of the material absorbing at 260 nm is RNA. Figure 25B shows the 260/230 ratio. A value of 1.0 or greater is considered a good index of purity. Figure 25 C shows the 260/280 nm ratio. A value of 2.0 is considered to indicate purity in the RNA.

PicoGreen and RiboGreen measurements of the DNA and RNA concentrations are shown in Table 25. Concentrations of DNA and RNA in the 100 uL of eluate are presented in Table 25. These values are presented in graphical form in Figure 26. In Figure 26A, DNA concentrations as measured by PicoGreen are shown. In Figure 26B, RNA concentrations as measured by RiboGreen are shown. The symbols used in Figure 26 are the same as those described in Figure 25. Note that greater binding and elution times do not translate into greater recovery of DNA and RNA. Also note that the use of NiFe₂C>4 particles results in recovery of considerably less RNA and slightly less DNA, see Figure 26 and Table 25.

Figure 27 shows DNA isolated using the various particles described in this example. The lanes in Figure 27 were loaded as follows: lane 0 - 2.5 uL Lonza Quantit markers; lanes 1-4, 30 minute elution of beads Co, Mn, Ni, and Zn composites of Fe204; lanes 5-8, 60 minute elutions, of beads Co, Mn, Ni, and Zn composites of Fe204; lanes 9-12, 90 minute elution of beads Co, Mn, Ni, and Zn composites of Fe204; lanes 13-16, 120 minute elution of beads Co, Mn, Ni, and Zn composites of Fe₂C>4. Arrows mark the position of the greater than 1500 base pair DNA and the material that migrates faster than the 100 base pair standard. Band densities from Scion Image are shown in Table 25. Graphical representations of these values are shown in panels A and B of Figure 29.

PCR results for amplification of the 451 base pair segment of the GAPDH gene are shown in Figure 28. Two images were taken during the electrophoresis process. During the early part of electrophoresis (top panel) there was considerable smearing around the 451 base pair amplicon. The bottom panel is the same gel at a later time in the electrophoretic process. As the samples move down the gel, the interfering material seems to separate from the amplicons. At this later time point, the 30 minute elution (lanes 1-4) seems to be darker than the 120 minute elution (lanes 13-16). The lanes of the gel in Figure 28 are the same as those in Figure 27, except Figure 28 also contains, in the side panel, lane 17 - negative control, and lane 18 - genomic DNA positive control.

Band densities from Scion Image for both the input DNA (Figure 27) and PCR amplicon (Figure 28) are shown in Figure 29. Numerical values of these band densities are given in Table 25.

Similar to non-magnetic NiO nanoparticles, NiFe₂C>4 magnetic nanoparticles deliver very little material that migrates faster than the 100 base pair standard. Much of this material might be small RNA given its abundance as measured by RiboGreen, see Table 25 and Figure 26B. The longer binding and elution times do not result in better PCR products, see Figure 28 and Figure 29 panel C. These data suggest that the extraction process may be made even faster. Optimal times for binding and elution still need to be established. Average values of all elution times for each particle type are shown in Table 25 as well as standard deviations are given in Table 26. There is a hint of particle type discrimination with various types of borate passivated MFe₂03. Row 2 of Table 25 lists the different M groups in the MFe₂C>3 magnetic nanoparticles used in this example.

Table 25 parameters used to compare different ferric oxides

Table 26 Mean + std for Table 1 values

NiFe₂0₄ magnetic nanoparticles performed the best for selectively isolating larger fragments of DNA from pooled normal human plasma following a dual protease digest. CoFe₂0₄ and MnFe₂0₄ performed the best for isolation of total RNA and material that migrates faster than the less than 100 base pair standard. It is interesting to note that for mass magnetization (emu/g) and relaxivity coefficient (mM^sec ¹), the values for MnFe₂0₄ are greater than for CoFe₂0₄ which are greater than for NiFe₂0₄. (See, Jun 2008). One explanation is that long pieces of DNA are required to cooperatively link NiFe₂0₄ nanoparticles. These results were generated with a permanent magnet from Spherotech (Lake Forest, Illinois) Finer tuning may be achievable with size (Jun 2008) and passivation alterations of the ionic double layer (Itri 2001). (Jun Y-W, Seo J-W, Cheo J (2008) Nanoscaling laws of magnetic nanoparticles and their applicabilities in biomedical sciences. Accounts of chemical research, 41(2) 179-189.) Example 20

In this example, a comparison was made among A1₂C>3, NiO, Ti0₂, and Ce0₂ particles. Only NiO consistently yielded DNA that worked well in the downstream application of PCR and did not yield significant amounts of smaller then 50 base pair material that could be DNA and small RNA. Table 27 Isoelectric points of nanoparticles

The isoelectric point (IEP) is the pH at which a particular molecule or surface carries no net electrical charge. Consideration of the isoelectric points (IEPs) of metal oxide ceramics may be useful in understanding the capture of nucleic acids at pH of about pH 4 to about pH 5. At pH values above the IEP, the predominate surface species is M-

O^" while at pH values below the IEP, M-OH2⁺ species predominate. IEP values except for NiO are from J. P. Brunelle, Pure and Appl. Chem., 50 (1978) 1211-1229.

In this example, 10 mM Tris pH 8 was added to the ethanol saline rinse that was originally implemented to remove protein and lipid. Tris was added in this step to remove electrostatically loosely attached shorter pieces of DNA and RNA.

This example was carried out as follows. Normal human plasma digest (prepared according to Example 14 was pooled for a volume of 1.5 mL. 0.54 g of urea was placed in a 2 mL polypropylene microfuge tube. Chipped away frozen BioReclammation plasma. Volume of plasma in urea was about 1.8 mL. 14 uL beta- ME and 45 uL

Savinase were added. Samples were digested for one hour at 52 C. The pH was adjusted to pH 4 with IN HCl for a final volume just over 2 mL. 20 mg pepsin from Amresco was added. Sample was digested at room temperature for 1 hour. Borate passivated nanoparticles were dispensed in replicates of four in 600 uL polypropylene tubes.

Volumes were normalized to optical density at 400nm (O.D 4oo_nm), e.g. 24 uL of AI2O3 with O.D 4oonm = 50. 125 uL of the 2 mL plasma dual protease digest was added and samples were vortexed. Samples were incubated at room temperature for one hour. Samples were centrifuged for 15 seconds in a Fisher 235C centrifuge. Pellets were rinsed twice with 500 uL 5 mM glycine, pH 3.3, and then rinsed twice with 200 uL 10 mM Tris pH 8, 75% EtOH v/v, 150 mM NaCl. Pellets in uncapped tubes were allowed to dry at 52°C for 30 minutes. DNA and RNA were eluted into 100 uL of 10 mM Na₂B₄0₇ pH 9 and 0.001 % Tween-20 for 30, 55, 75, and 95 minutes at 52°C. 9 uL of the eluted material was diluted with 81 uL of 10 mM Tris pH 8 for absorbance measurements. 30 uL of diluents were mixed 1 : 1 with PicoGreen and RiboGreen working stocks to measure the concentrations of DNA and RNA, respectively. GAPDH PCR was performed with 5 uL of sample in a total reaction volume of 20 uL.

UV absorbance results are shown in Table 28. Of the two particles having IEP greater than 7, see Table 27, NiO yielded the product with the best 260/280 ratio suggesting less protein contamination. Two tail t-tests were used to compare NiO with the other particles. No elution time dependence for any of these parameters was seen (not shown). Results in Table 28 further establish NiO as having the most optimal 260/280 ratio though the difference with ceria (Ce0₂) was not significant at the p<0.05 level. PicoGreen and RiboGreen measurements of RNA and DNA are shown in Table 2.

Table 28 UV Absorption

Table 29 PicoGreen and RiboGreen measurements of RNA and DNA

PCR input DNA is shown in Figure 30. The lanes in the gel shown in Figure 1 were loaded as follows: lane 0 - Lonza Quantit standards; lanes 1-4 - AI2O₃ 30-95 minute elutions; lanes 5-8 - Ce0₂ 30-95 minute elutions; lanes 9-12 - NiO 30-95 minute elutions; lanes 13-16 - Zr0₂ 30-95 minute elutions. Results of the GAPDH PCR are shown in Figure 31. The lanes in Figure 31 are the same as those in Figure 30 with the addition a (-) lane containing H₂0 as a negative control and a (+) lane containing genomic DNA as a positive control. Scion Image band density values in arbitrary units are given in Table 30. Note that all four particle types yield about the same amount of greater than 1500 base pair material, see Table 30. The amount of less than lOObp material is considerably greater in the samples isolated with alumina particles than in those isolated with NiO. PCR was performed with GAPDH PCR primers. Results are shown in Figure 31. All of the NiO isolated samples (lanes 9-12) yielded the GAPDH product. Only faint PCR product could be detected from the samples isolated with zirconia (lanes 13-16). In this particular example, PCR product was obtained from material isolated with alumina that was significantly less intense than that obtained with NiO nanop articles, Table 30. There seems to be a rank order, NiO, Ce0₂, AI2O₃, Zr0₂, in the particles that gives the strongest greater than 1500 base pair band and the best PCR results, see Table 30.

Table 30 Scion Image band density data

In this example, there does not appear to be a correlation between IEP and the amount of DNA or RNA isolated. Similarly, there was no correlation between IEP and the consistency of PCR product. Alumina produced the best results for isolating the most total RNA and DNA. NiO produced the best results for isolating only the greater than 1500 base pair DNA, and NiO yielded the most consistent PCR product. These results illustrate that the chemical differences in the particles can be used to selectively enhance yields depending on the desired outcome.

Example 21

This example compares four highly basic particles with different crystal structures. NiO and MgO have a cubic structure, and La₂0₃ and Al₂0₃ have a hexagonal structure. Particles were used in the unpassivated state. Only the DNA isolated using NiO and MgO yielded DNA that gave a PCR product and little material smaller than 50- 100 base pairs. Table 31 Physical properties of nanoparticles used

The isoelectric point (IEP) is the pH at which a particular molecule or surface carries no net electrical charge. Consideration of the isoelectric points (IEPs) of the metal oxide ceramics used is helpful in understanding capture of nucleic acids at pH 4 to pH 5. In Example 16, Glycine was explored as a passivating agent. In this example, no passivating agent was used, as the particles themselves may change the pH of the digest to which they are added. In this example, different crystal geometries of the particles are compared.

This example was carried out as follows. AI2O₃ was purchased from Sigma. All other nanoparticles were supplied by Inframat Advanced Materials. Because La₂C>3 and MgO are soluble at acidic pH, all four varieties of nanoparticles were used in their unpassivated state. 1-2 mg of each of the four types of nanoparticles were dispensed into 600 uL microfuge tubes in replicates of four. 250 uL of a dual protease pooled normal human plasma digest prepared in the same manner as Example 14 was added to replicates 1-3. To the 4^th replicates, 350 uL was added. After 1 hour at room temperature and periodic vortexing, samples were centrifuged for 20 seconds. The supernatant was discarded. Removed residual supernatant in the 600 uL tube with alOO uL pipette tip. Samples were rinsed once with 500 uL 5 mM glycine, pH 3.3, pipetted up and down twice, and centrifuged for 20 seconds. The supernatant was discarded. Removed residual supernatant in the 600 uL tube with 100 uL pipette tip. Repeated glycine rinse for a total of two glycine rinses. Samples were rinsed with 500 uL of the lOmM Tris pH 8 buffered EtOH/saline rinse. Samples were evaporated at 52°C for 15 minutes. Replicates 1-3 were resuspended in 100 uL of 10 mM Na₂B₄0₇ pH 9 and 0.001% Tween-20. To each of the number 4 replicates, 140 uL of the same solution was added to complensate for the larger starting volume.

Standard GAPDH PCR was performed with 5 uL of sample in a reaction volume of 20 uL. Pico Green and Ribo Green measurements of DNA and RNA concentrations were performed on 3 uL of sample diluted with 27 uL lOmM Tris pH 8. These diluents were mixed 1:1 with PicoGreen and RiboGreen working stocks. For measurements, the spectrophotometer was zeroed with the elution buffer. Absorbance was measured without dilution.

Table 32 UV Absorbance

UV absorbance data is shown in Table 32. Since MgO yielded the best PCR results (see Figure 33), all particles were compared to MgO using a two tailed paired t test. Even with the EtOH/saline/Tris pH 8 rinsing that was designed to remove smaller nucleic acids that were bound with less cooperativity than longer sequences, the yield of RNA is still about what has traditionally been observed.

Table 33 PicoGreen and RiboGreen readings of DNA and RNA concentrations

The amount of DNA isolated from the AI2O₃ and L_a20₃ particles is significantly greater than that isolated from the MgO particles, see Table 33. The scant amount of

DNA greater than 10,000 bsae pairs is only slightly greater in the La₂0₃ isolated samples than in the MgO isolated samples, see Table 34. Contrast this to total DNA in Table 33. The difference lies in the amount of nucleic acid that migrates faster than the 50 base pah- standard, see Table 34 and Figure 32A. NiO and MgO appear to be isolating less total DNA but are selecting for the larger fragments.

Figure 32 is a gel of DNA that was isolated according to this example and that was used as input DNA for a GAPDH PCR reaction. Figure 32A shows DNA and RNA migrating faster than the 50 base pair standard. Figure 32B shows DNA greater than 10,000 base pairs. The larger band density is about the same for all particle types but, the faster than 50 base pair material is noticeably different among particle types. The lanes in Figure 32 were loaded as follows: lane 0 - Sigma wide range direct load markers; lanes 1- 4 - A1203 samples; lanes 5-8 - La203 samples; lanes 9-12 - MgO samples, lanes 13-16 - ZiO samples.

Figure 33 is a gel of the PCR products from the GAPDH PCR using DNA isolated in this example. The lanes of the gel in Figure 33 are the same as those in Figure 32 with the addition of lane 17 - water as a negative control; and lane 18 - genomic DNA as a positive control. DNA isolated with MgO and NiO particles gave the best PCR products, see Figure 33 and Table 34.

Table 34 Gel band densities from Scion Image

All of the particles surveyed in this example have high IEP. AI2O₃ and La₂0₃ particles yielded more DNA and RNA. The better yield seemed to be represented by material that migrated faster than the 50 base pair DNA standard. This material seems to interfere with PCR. NiO and MgO particles with the cubic geometry appear to isolate only the greater than 1500 base pair species of DNA. Passivating the particles appears to buffer the metal oxide groups that are alkali in this example. Lack of passivation did not prevent DNA/RNA capture.

Claims

A method of isolating nucleic acids from a biological sample comprising:

a) providing a biological sample

b) performing a digestion under conditions at a pH of between about pH 3.5 to about pH 7.0 and a temperature between about 5°C to about 40°C, wherein the pH and the temperature are balanced to minimize depurination of the nucleic acid.

The method of claim 1 wherein a chaotropic agent is added to the digestion. The method of claim 2 wherein the chaotropic agent is urea or guanidine HC1. The method of claim 1 wherein the digestion is performed with pepsin. The method of claim 1 wherein a reducing agent is added to the digestion.

6. The method of claim 3 wherein the chaotropic agent is urea and the digestion is performed with pepsin.

A method of isolating nucleic acids from a biological sample comprising:

a) providing a biological sample

b) performing a first digestion under alkaline conditions at a pH between about pH 7 and about pH 9, and a temperature between about 20°C to about 60°C;

c) performing a second digestion under conditions at a pH of between about pH 3.5 to about pH 7.0 and a temperature between about 5°C to about 40°C, wherein the pH and the temperature are balanced to minimize depurination of the nucleic acid.

The method of claim 7 wherein a chaotropic agent is added to the first digestion.

9. The method of claim 7 wherein a reducing agent is added to the first digestion.

10. The method of claim 7 wherein the first digestion is performed with Savanase and Protease K. 11. The method of claim 7 further comprising an alcohol precipitation step is added between the first digestion and the second digestion.

12. The method of claim 7 further comprising separating the nucleic acids from the using metals, metal oxides, ceramics, or combinations thereof.

13. The method of claim 12 wherein the metals, metal oxides, and ceramics are selected by matching the affinity of the isolectric point of various metals, metal oxides, and ceramics to the nucleic acid at the pH of the digestion. 14. The method of claim 12 wherein the metals, metal oxides, ceramics, or combinations thereof further comprise particles, nanop articles, beads, fibers, mesh, screens, or combinations thereof.

15. The method of claim 12 wherein the metals, metal oxides, ceramics, or combinations thereof further comprise magnetic beads.

16. The method of claim 12 wherein the metals, metal oxides, ceramics, or combinations thereof remain in colloidal suspension in aqueous solution. 17. The method of claim 15 wherein the magnetic beads remain in colloidal suspension in aqueous solution.

18. The method of claim 13 wherein the metals, metal oxides, and ceramics are selected to selectively isolate RNA over DNA.

19. The method of claim 13 wherein the metals, metal oxides, and ceramics are selected to selectively isolate DNA over RNA.

20. The method of claim 13 wherein the metals, metal oxides, and ceramics are selected to selectively isolate a predetermined size range of nucleic acids.