+++, excellent; ++, very favorable; +, moderate; —, less favorable. *If freeze-dried, rating becomes ++.

+++, excellent; ++, very favorable; +, moderate; —, less favorable. *If freeze-dried, rating becomes ++.

which it is to be used and that all of its physical and chemical properties are fully documented.

2. How versatile is the material? i.e., is it technically suitable for use in all the possible testing methodologies?

As indicated above, a wide range of molecular genetic techniques can be used to test for the same mutation. Some forms of RM will be suitable for a wider range of tests than others. In essence, the more like total genomic DNA a RM is the more versatile it will be, so this parameter is linked to the previous one. Reference materials based on short DNA fragments, whether cloned, produced by PCR, or chemically synthesized will not be useful RMs for tests which involve Southern blotting or other longrange techniques, but they may be excellent RMs for short-range techniques. The testing methodologies employed are tailored to the type of mutation under study: short-range techniques for point mutations and long-range techniques for large triplet repeat expansions and major gene rearrangements. Thus the optimal type of RM will depend on what type of mutation the RM is designed for. When RMs for highly multiplexed tests are required, only artificial RMs will normally be possible, as natural sources will only contain at most two mutations.

3. How stable is the material likely to be?

As a batch of RMs must have a reasonable shelf life to be useful (5 years is a sensible target), the stability of the proposed RM is of central importance. Experience of maintaining DNA banks in diagnostic and research centers tells us that purified DNA is one of the more stable biomolecules, and may survive intact in solution at 4°C or even room temperature for many years, as long as it is stored in a suitable solution and at high concentration. Freeze-dried DNA may be stable at room temperature.

Intact cells are likely to be the least stable of all the forms of RM, as they need to be preserved at at least — 70°C and preferably in liquid nitrogen to ensure their integrity. The other RM types, all consisting of purified DNA, may have similar stability characteristics, but this will need to be determined experimentally. The very large fragments in purified genomic DNA are more likely to be affected by physical shearing on storage in liquid, or on freezing and thawing, than the shorter fragments in PCR products or recombinant DNA. Dried blood in spots appears to be reasonably stable over several years at room temperature.

4. How expensive or economical is it to produce large batches of the material?

In order to meet the requirements of a certified reference material, the material must be capable of being prepared in large batches suitable for many hundreds or even thousands of tests. This is possible for all of the RM types mentioned above, but is likely to be much more economical in the cases of PCR products and cloned DNA fragments. A 20 pL PCR reaction, diluted one million-fold for use as an RM, yields 20 liters of the product.

5. How expensive or economical is it to store and to transport the material?

With RMs for molecular genetic testing, we are dealing with very small quantities of material in each aliquot, so bulk is not a problem to be considered. The parameters contributing to the cost of storage and transport are stability (discussed above) and hazard. Storage and transport costs will of course rise sharply with decreasing stability because of the requirement for refrigeration/freezing. Hazards associated with biological materials include pathogens which may be harbored by cell lines but most potential contaminants are unlikely to be present in purified DNA.

6. Are there ethical or ownership issues associated with the material?

It is clear that apart from synthetic DNA, which is the least suitable material from other points of view, all types of RM are derived directly or indirectly from a human source. Full ethical approval must be obtained from donors of material for the establishment of cell lines, or the use of blood or other samples in RM development. Concerns may remain, however, that consent may be later withdrawn or challenged, or that testing of the RM for mutations other than the one specified in the certification process might constitute an invasion of the donor's genetic privacy. Furthermore, materials derived from human sources may be the subject of ownership claims when used for commercial purposes. Thus the further removed from a human source an RM is the less likelihood there is that such problems may arise. Cloned DNA fragments are removed from their genomic context, but their derivation must be documented under Genetically Modified Organism (GMO) regulations. Polymerase chain reaction products must be derived from a genomic DNA sample, which is traceable to an individual.

The approach must then be to minimize the potential consent and ownership problems by obtaining appropriate informed consent and releases for each human source. Other legal issues concern transportation of RMs over boundaries. Often, a human- or biological-source-derived material is deemed to be potentially hazardous, which may hinder distribution of RMs in international initiatives.

Some types of reference material and their suitability under the headings discussed are summarized in Table 1.

and validate new reference materials for genetic testing[4] which are ongoing both in academic centers and in the private sector.[5]

Ongoing Initiatives to Develop Reference Materials

Several initiatives are ongoing to produce RMs for genetic testing in Europe and North America. In Europe, the CRMGEN project, funded by the European Union's Fifth Framework for Research, is developing reference materials in the form of cell lines, genomic DNA, PCR products, and plasmids for a range of common genetic tests ( Based on the expertise gained in the project, guidelines for the future production and validation of RMs will also be developed and published. The European Commission's Institute for Reference Materials and Measurements (IRMM) is collaborating with the International Federation of Clinical Chemistry (IFCC) to produce plasmid-based RMs for prothrombin variants. In the United Kingdom, the National Institute for Biological Standards and Control (NIBSC) is working on a number of gDNA RMs for Factor V Leiden testing based on cell lines.

In the United States, the Centers for Disease Control and Prevention are coordinating efforts to source, produce,

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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