Wednesday, 01 October 2014
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Scientists have redeveloped HDPE for use in biochemical applications such as DNA analysis.

High-density polyethylene (HDPE) might not appear to be a very advanced high-technology material. Indeed, at Wrexham-based Porvair, the thermoplastic has been widely used for years to manufacture sintered porous materials for diverse applications in filtration, aeration, silencing and perfume emanation.

But when chartered with the goal of finding new uses for the material, Dr David Cowieson, the specialist filtration-technology group’s product development manager, realised that, by chemically functionalising its surface, it could be tailored for use in a number of new biochemical applications.

HDPE disks can be employed in a system that uses ChIP assays

HDPE disks can be employed in a system that uses ChIP assays

Fortuitously, through the Welsh Assembly government’s XGEN consortium of advanced manufacturing companies, Cowieson was introduced to Dr Steve Conlan, director of the Centre for Nanohealth at Swansea University, who appreciated that such modified HDPE disks could be employed in a system that performs a highly specific function within the Chromatin Immunoprecipitation (ChIP) assay.

Over the past 12 years, such ChIP assays have become an increasingly important method by which researchers can analyse interactions between cellular Deoxyribonucleic Acid (DNA) and proteins.

The assay process itself involves cross-linking DNA to proteins that interact with the DNA in live cells by treating the cells with formaldehyde. This chromatin is then sheared to smaller fragments by ultrasound in a process known as sonication. Next, the protein-bound DNA fragments are pulled out of the solution by immunoprecipitation, after which a polymerase chain reaction (PCR) is used to determine how much DNA associated with a specific protein has been recovered.

The process can be used to isolate a particular protein from a sample containing many different types

’As a result of discovering which parts of a cell’s DNA act together with any given protein, researchers can gain a greater understanding of how the consequent biochemical reactions that arise might be responsible for causing diseases such as cancer,’ explained Swansea University’s Dr Conlan.

During the immunoprecipitation step of the ChIP process, the protein-DNA fragment is brought out of a solution using an antibody that specifically binds to that particular protein. The process can be used to isolate a particular protein from a sample containing many thousands of different types.

In the past, researchers have used a number of technologies to perform the immunoprecipitation step of the assay. One of the most commonly used techniques is to immobilise antibodies on either microscopic magnetic or non-magnetic beads coated with a protein called Protein A, which was originally found in the cell wall of the bacteria Staphylococcus aureus.

’In this process, antibodies are added to a DNA-protein mixture that has been extracted from a sample of human tissue. The proteins that are targeted by the antibodies are then captured onto the beads, after which they are separated magnetically or in a centrifuge,’ said Dr Conlan.
However, both of these methods have their drawbacks. While they do perform effectively, they both require a relatively large sample size, are awkward to manipulate and some loss of the beads is inevitable, leading to a reduced amount of protein recovered in the process.
Now, through the collaborative

research effort between Porvair and the Centre for Nanohealth, an alternative process of obtaining the proteins has been developed and characterised that could make the separation procedure faster and more robust.

The procedure makes use of the same principle used by the former bead technology, in that Protein A is again used to bind to the antibody-bound proteins of interest. But rather than using beads as the platform for the protein, the system makes use of the company’s porous polyethylene discs. But first, they needed to be functionalised.

’To manufacture the modified high-density polyethylene [HDPE] disks specifically for use in a ChIP system, the inert hydrocarbon material is first chemically oxid

Chromatrap kits will offer protein-impregnated disks bundled in small plastic columns

Chromatrap kits will offer protein-impregnated disks bundled in small plastic columns

ised, making its surface reactive. Short-chain hydrocarbons are attached to the surface that are then bound to the larger Protein A molecules,’ explained Cowieson.

The disks are then assembled into a column through which the mixture of sonicated Chromatin flows. The Protein A on the disks then binds to antibody-tagged protein-DNA fragments as the remaining solution passes through to a container beneath the columns.

The columns of plastic disks are then washed several times to ensure that there is no trace of contaminants. Lastly, a release reagent is passed through the column, which breaks the Protein A chromatin bond, leaving a solution containing only the Chromatin complex that can be analysed following further biochemical manipulation.

’The technique has several advantages over its rivals. Because the impregnated disks are not loose particles like beads, they cannot move within the liquid. That means that there is no requirement for either a centrifuge or a magnetic separation system to extract the Chromatin complex from the solution. The solution flows through the disks within the column and the complex is captured on the surface,’ said Cowieson.

Conlan’s team, which is working on ways of speeding up the diagnosis and treatment of diseases such as cancer, has been characterising the performance of the Porvair technology to determine its effectiveness as a means of immunoprecipitation.

After conducting a lengthy analysis, Dr Conlan and his team noted that the disk-based procedure was not only faster than the commonly used bead-based technology, but also allowed his researchers to perform assay tests on more samples at once.

“When analysing the sample there is less background noise caused by the amplification of irrelevant signals”

DAVID COWIESON, PORVAIR

Dr Conlan also said that the disk-based system provided a better signal-to-noise ratio too meaning that, in the future, a smaller sample could be taken from a patient during a biopsy procedure prior to analysis through the assay process.

’This improved signal-to-noise ratio is due to the fact that the disk-based technology does not bring out of a solution as many non-specific protein-DNA fragments in addition to the ones of interest to the researcher. Because of that fact, when the polymerise chain reaction is performed to analyse the sample, there is less background noise caused by the amplification of irrelevant signals,’ said Cowieson.

Conlan said that he would be particularly interested in using the technology as part of his own research examining the specific interactions between certain proteins and DNA that may be responsible for causing endometrial cancer. Those proteins, which control the regulation of genes that reside on discreet stretches of DNA, are a critical component in the causes of certain disease pathways.

In the meantime, he is working with the Porvair chemists to characterise the specific sensitivity, level of background binding and repeatability characteristics of the technology so that the Porvair team will be able to benchmark the system against the competing technologies before launching it onto the market.

Porvair’s product development plans are now well forward. Cowieson said that his team at Porvair will produce products based on the disk technology. Named Chromatrap, these will take the form of a kit, or a group of kits, in which the protein-impregnated disks will be bundled in small plastic columns. The kits would also contain all the reagents that are needed to perform the immunoprecipitation procedure.


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