Preparation and Validation of PCR-generated Positive Controls for Diagnostic Dot Blotting

¹ Molecular Diagnostic Laboratory, Institute for Molecular and Human Genetics, Georgetown University, Washington, DC 20007;
² Dept. of Pathology and Pediatrics, Childrens Hospital, Los Angeles, CA 90027;
^a author for correspondence: Molecular Diagnostic Laboratory, Institute for Molecular and Human Genetics, 3800 Reservoir Rd., NW, Rm M4000, Georgetown University Medical Center, Washington, DC 20007, fax 202-784-1770, e-mail wonglj{at}gunet.georgetown.edu

Allele-specific oligonucleotides (ASOs) are small, single-stranded nucleotide polymers (~18–20 bases in length) of diagnostic utility given their ability to hybridize to single-stranded DNA target molecules in a sequence-specific, temperature-dependent manner. Accordingly, two target molecules differing in composition by a single base can be distinguished by hybridization with an ASO that is complementary to one of the target molecules but noncomplementary to the other by a single base. The latter target produces a single basepair-mismatched double strand that dissociates at a lower washing temperature than the fully complementary, double-stranded molecule.

Since first used to screen for beta-S globin mutations in sickle cell disease, ASOs have been widely used to detect known disease-causing mutations and disease-associated base substitutions (1)(2)(3). Diagnostic screening using ASOs for clinically relevant base substitutions (mutations/polymorphisms) frequently uses a dot blot format. PCR-amplified patient DNA is bound to a membrane, denatured to generate single-strand targets, and probed with both mutant and wild-type ASOs. Subsequent washing under optimized conditions denatures all but the entirely complementary double-stranded hybrids, which yield a positive signal. Dot blotting requires positive-control samples to monitor the sensitivity and specificity of each diagnostic blot. Given the rarity of some disease alleles, the acquisition of positive-control DNA is often rate-limiting in the establishment of a dot blot protocol for a given disease.

Mitochondrial disease phenotypes have been associated with an array of base substitutions in the mitochondrial genome, and diagnostic screening with ASOs is an effective approach to the identification of mitochondrial DNA mutations. For example, an adenine-to-guanine transversion at position 4136 (A4136G) of the human mitochondrial genome, which leads to the substitution of cysteine for tyrosine in the ND1 subunit of respiratory chain complex I, has been associated with Leber hereditary optic neuropathy (4)(5)(6). Using the A4136G substitution associated with Leber hereditary optic neuropathy as a prototype, we have validated a technical approach to the preparation of synthetic positive controls suitable for ASO dot blot analysis in any disease.

The approach involves generating PCR products containing the base substitution of interest by using a single-basepair-mismatched PCR primer to amplify normal target DNA. This produces abundant mutation-containing PCR product (synthetic, positive-control DNA). The integrity of this product as positive-control DNA for the purposes of dot blotting was validated by probing a PCR product from a patient known to carry the A4136G base substitution in parallel dot blotting experiments.

Four PCR primers were used (Table 1 ) to generate three PCR products. The primer pair Mt-1/4508R and template DNA from a patient known to carry the A4136G base substitution were used to make a 1379-bp product with nucleotide position 4136 located >300 bp from one end. A 1016-bp product was prepared using the primer pair Mt-1/A4136nl and a wild-type DNA template that contained the wild-type base adenine at nucleotide position 4136. The primer pair Mt-1/A4136G, which includes a single-basepair-mismatched reverse primer (A4136G), and normal template DNA were used to generate the synthetic positive-control PCR product: a 1016-bp product containing a guanine at nucleotide position 4136. Note that in each of the 1016-bp products, nucleotide position 4136 lies within 10 bp of one end of the PCR product. Appropriate and otherwise identical reactions without templates (water controls) were also run.

The PCR conditions were standard (2). The quality of amplification was monitored by agarose gel electrophoresis. The sequence of each of the four PCR products was confirmed by automated sequencing using a Taq Dye Deoxy terminator cycle sequencing protocol (Applied Biosystems) with MT4013F (5'-CCCTCACCACTACAATCTT-3') serving as a sequencing primer. Duplicate dot blots for hybridization with either normal or mutant probes were performed as described previously (2). The results are shown in Fig. 1 . The ASO A4136nl yielded a positive result for the wild-type PCR product generated with the primer pair MT-1/A4136nl (lane E). The ASO A4136G yielded a positive signal of equal strength and intensity for both the known A4136G-containing DNA sample (lane A) and the synthetic PCR-generated positive-control sample (lane C). All water controls (lanes B, D, and F) were negative.

View larger version (21K): [in this window] [in a new window]   Figure 1. ASO dot blot of synthetic and authentic positive controls. The PCR reactions were described in the text. The forward primer in all six reactions was mt-1. Lanes B, D, and F, water (no template) controls for lanes A, C, and E, respectively. Lane A, authentic positive control: A4136G-containing DNA template with 4508R reverse primer; lane C, synthetic positive control: wild-type DNA template with A4136G reverse primer; and lane E, normal control: wild-type DNA template with A4136nl reverse primer. The upper strip was hybridized with the normal probe A4136, and the lower strip was hybridized with the mutant probe A4136G.

These side-by-side blots validate the use of synthetically generated PCR products with a base substitution as positive controls for ASO/dot blotting protocols. Compared with naturally occurring alleles that contain base substitutions, the synthetic mutant products performed with the same level of specificity and sensitivity. The synthetic control contained the substituted base within 10 bp of the end of the PCR product, compared with the naturally occurring PCR product in which the substituted nucleotide was located >300 bp from one end. This did not adversely affect the quality of the signal.

The effectiveness and more general applicability of this approach is further supported by our success in generating synthetic positive-control samples for several other mitochondrial base substitutions, including A3243G, T3290C, C8359A (sequence confirmed), C3212T, T4216C, T4160C, T4336C, T3250C, C3256T, C3303T, A3252G, A4269G, A3397G, A3251G, A3260G, A3302G, T8851C, T9176C, T12311C, T14484C, T14709G, G15812A, A12308G, A15923G, and A15924G. In addition, we have also succeeded in preparing synthetic positive controls for the cystic fibrosis transmembrane regulator (CFTR) gene base substitutions: T2872C (sequence confirmed), C583T, A425G, A698G, and A1968C. It should be emphasized that the sequence of the newly synthesized positive controls should be confirmed either by direct sequencing or by testing with the appropriate authentic positive-control DNA. Otherwise, if there is a mistake in the oligonucleotide sequence, use of the same oligonucleotide for the PCR primer and the dot blot probe could generate an undesired positive control result and a false-negative patient result.

Acknowledgments

D Johnson was supported by the Las Madrinas Endowment for Molecular Genetics at Childrens Hospital, Los Angeles, CA.

Footnotes

¹ present address: Yale University, School of Medicine, New Haven, CT 06520-8066

References

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