Generating copy number standards

PCR generated DNA templates: by using a pair of nested primers it is quick and easy to generate and quantitate a template to act as a standard for your reactions. The first primer pair generates a large PCR product, containing the region of mtDNA for investigation. This is then purified before being quantified. The second nested primer pair is used for the real-time reaction. We have found from experience that there are some regions that simple will not perform well as real-time templates. This is probably due to sequence characteristics leading to unwanted secondary structure within the PCR product.

There are significant differences between the available methods for PCR product purification and the subsequent performance of these products as real-time PCR templates. PCR purification spin columns are one quick and easy method for removing salt and unused PCR components from a product. However, we have found that despite using no buffers, these columns have a severely detrimental effect on real-time performance (Figure 11.3B). It may be

Figure 11.3

Different template preparations behave differently when used as absolute quantification standard curves for two mtDNA assays. Reaction One detects wild-type using a region of ND4 (black), the second detecting total mtDNA using a region of ND1 (grey). Each graph shows the standard curve for both reactions performed on 1:10 serial dilutions from different templates. A. whole genomic DNA extract from blood; B. PCR amplified product, purified using PCR purification spin columns; C. PCR amplified product, purified using gel extraction; D. PCR product cloned into vector, circular; (E) PCR product cloned into vector, linearized.

through an interaction with the spin column membrane, mechanical disruption or some chemical leeching from the membrane into the purified DNA sample. We have routinely used spin columns to purify PCR products for DNA sequencing with no problems, and continue to do so. However, we have been unable to use this technology to purify templates for real-time PCR reactions.

For the purification of PCR templates to be used as real-time standards we would recommend the use of a gel extraction method. This process removes all unincorporated PCR reaction components and leaves a single clean template for use as a standard. However, when compared to genomic DNA dilution series PCR template reactions do not always perform the same (Figure 11.3C) and so caution is advised.

Cloned PCR templates: to ensure a continuing and reproducible supply of your template it is convenient to use the nested primer pair approach, with the addition of cloning the primary, large product into a vector before transforming it into competent cells, extracting and purifying the DNA. This has the added advantage of ensuring you have a homoplasmic supply of your target region. For rare or low level heteroplasmic mutations this may be of particular interest. Two lines of thought exist regarding the use of cloned PCR products as real-time templates. The first suggests that for maximum PCR efficiency the vector needs to be linearized by restriction digest prior to real-time amplification. The second is that the vector be used in its closed circular form. We have investigated both variations and compared to the data obtained from genomic mtDNA. We have found that for mitochondrial studies, reactions behave differently on cloned templates, both closed (Figure 11.3D) and linear (Figure 11.3E), than on genomic mtDNA.

Genomic mtDNA: there is something reassuring about being able to compare like with like. Copy number standard curves based on PCR products are not the same as 16.5 kb closed circular mtDNA genome molecules (Figure 11.3A). However, there is the difficulty of quantifying mtDNA in a DNA extract due to the presence of nDNA. We have investigated several approaches to this. Ultra-centrifugation through a caesium chloride gradient enables the separation of mtDNA from nDNA by the differential movement through the gradient. We have found that through using nDNA specific primers a product can be amplified from the 'clean' mtDNA fraction.

Southern blot analysis is another approach that could be used to quantify mtDNA in a DNA extract. By running a known set of standards, quantified pure PCR products, along with several dilutions of the homogenate DNA sample and probing with an mtDNA specific probe the quantity of mtDNA can be calculated. Quantification through this approach should be viewed as inaccurate, in our opinion. There appears to be preferential binding of the probe to the purified PCR products. This may be due to the PCR products being short linear molecules that the probe can access more readily.

The third approach requires a reliable real-time assay to already be in place using one of the other two methods of generating a standard curve. A genomic DNA sample can be quantified by real-time using either a PCR template or cloned PCR product standard curve. Experience has shown that for difficult amplicons the option of PCR template or cloned product as a standard is not viable. However, other regions prove to be remarkably reproducible by these methods. Awareness of the potential to introduce errors in the calculation is inherent with this approach. Back calculation of the quantification by independent methods is advised, either spectropho-tometry or fluorimetry. Quantification of a genomic mtDNA sample by this template real-time route has great benefits when investigating a difficult region. A second benefit to this is the provision of a quantified standard for any mitochondrial assays you design in the future. An example of absolute quantification in determination of viral load is given in Chapter 14, Protocol 3.

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