Janet Rettig Emanuel

PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR

PCR has become a powerful tool for genetic analysis and many applications for gene sequence quantitation are based on this technology ( 1–3). Standardized reaction conditions require accurate quantitation of input DNA as well as optimization of chemical and cycling parameters. In this study we discuss PicoGreen (Molecular Probes, Eugene, OR) fluorescence enhancement as a useful assay for template DNA quantitation and PCR product formation. Spectrophotometry is the principal method for evaluating quantity and quality of nucleic acids. In aqueous solution, DNA has maximal absorbance near 260 nm with an extinction coefficient of 50; protein absorbs light strongly near 280 nm. The concentration of a sample can be read directly (in μg/μl) by diluting it 1:20 in water or buffer; a practical lower limit of detection is 50–100 ng DNA in a 50–100 μl microcuvette. The A260/A280 ratio provides an estimate of DNA purity; values of 1.7–2.0 predict ‘clean DNA’. However, single-stranded DNA, RNA, PCR primers and dNTPs, or aromatic organic compounds such as phenol interfere by absorbing light in this range. Fixed tissue samples with substantial protein crosslinking and DNA preparations containing added enzymes or protein stabilizers are difficult to evaluate spectrophotometrically ( 4). Intercalating fluorochromes, such as ethidium bromide or Hoechst 33258, selectively bind to dsDNA. The sensitivity of Hoechst 33258 is ∼25 ng of DNA per assay, but preferential association with domains of high A–T content or reduced binding to DNA fragments <500 bp may result in skewed analysis ( 5). Accurate evaluation may require sophisticated or dedicated equipment since both dyes photobleach easily and fluorescence enhancement of DNA binding is low, leading to high background readings. These compounds are carcinogenic and pose handling and disposal problems. Electrophoretic array is the most common means of evaluating molecular distribution of both simple and complex DNA samples. When stained with ethidium bromide, transillumination with 254 nm UV light permits CCD camera visualization of a single agarose gel band containing ∼5 ng or a polydisperse sample containing 25–50 ng of dsDNA. SYBR-Green I  (Molecular Probes, Eugene, OR) is a proprietary fluorescent dsDNA-specific stain that has an emission peak at 520 nm following excitation at 254 or 497 nm. Image collection and analysis with 254 nm transillumination requires the use of an optical quality band-pass filter to eliminate infrared interference. SYBR-Green I is more sensitive than ethidium bromide with a limit detection of ∼50 pg per band or ∼250 pg per lane polydisperse dsDNA. Argon la er-activated gel scanning or capillary electrophoresis is more sensitive ( 6), but far more costly. Gel analysis allows evaluation of genomic DNA integrity, completeness of restriction endonuclease digestion and quantity of late cycle PCR products. However, this method is impractical for routine or high throughput DNA quantitation ( 7). PicoGreen is a fluorochrome that selectively binds dsDNA and has characteristics similar to that of SYBR-Green I. It has an excitation maximum at 480 nm (lesser peaks in the short-wave UV range) and an emission peak at 520 nm. When bound to dsDNA, fluorescence enhancement of PicoGreen is exceptionally high; little background occurs since the unbound dye has virtually no fluorescence. PicoGreen is very stable to photobleaching, allowing longer exposure times and assay flexibility. However, the molecular structure of the dye is proprietary and the mode of binding is not fully characterized, so potential handling hazard is unknown. We evaluated PicoGreen for quantitation of multiple DNA sample types. We examined the linearity of binding and the effective detection range for different species of ‘high molecular weight’ DNA standards (human placental, calf thymus and λ phage; with or without restriction digestion) and DNA isolated from a variety of tissue types preserved under different protocols. We also assayed ‘low molecular weight’ dsDNA (∼150 bp PCR products) in the presence or absence of reaction primers, dNTPs and Taq polymerase. Oligonucleotide primers were evaluated for interference with quantitation in some samples. Control DNAs were from commercial sources. EcoRI (New England Biolabs, Beverly, MA) digests were performed with 5 U per sample in a 10 μl reaction mix at 37 C for 2 h. We obtained sample DNA by organic extraction from flash-frozen or paraformaldehyde-fixed paraffin-embedded surgical remainder tissues. PCR mixtures contained 0.2 μM each primer, 50 μM each dNTP, 0.02 U/ μl AmpliTaq polymerase (Perkin-Elmer Corp., Wilton, CT) and TaqStart MAb (Clontech, Palo Alto, CA). Primers were removed from PCR reactions with Microcon 30 (Amicon, Beverly, MA). The A260/A280 of each sample was read against a TE blank in a Lambda-2 Spectrophotometer (Perkin Elmer Corp., Norwalk, CT), fitted with a 100 μl quartz microcuvette. DNA samples were diluted 1 μl into 100 μl of TE. A reading of 0.020, the lower confidence level of the instrument, represented 100 ng of DNA

Rat-brain Na,K-ATPase beta-chain gene: primary structure, tissue-specific expression, and amplification in ouabain-resistant HeLa C+ cells.

We deduced the complete amino acid sequence of the rat brain Na,K-ATPase beta-subunit from cDNA. The rat brain beta-subunit exhibits a high degree of primary sequence and secondary structural homology with the human and Torpedo beta-subunit polypeptides. Analysis of rat tissue RNA reveals that the beta-subunit gene encodes four separate mRNA species which are expressed in a tissue-specific fashion. In ouabain-resistant HeLa C+ cells, beta-subunit DNA sequences are amplified (approximately 20-fold) and beta-subunit mRNAs are overproduced relative to levels in parental HeLa cells. These results suggest that the beta-subunit plays an important role in Na,K-ATPase structure-function and in the mechanism underlying cellular resistance to the cardiac glycosides.

Detection of thyroglobulin, thyroid peroxidase, and RET/PTC1 mRNA transcripts in the peripheral blood of patients with thyroid disease.

PURPOSE Detection of mRNA transcripts for thyroglobulin (TG), thyroid peroxidase (TPO) and RET/PTC1 in the peripheral blood of patients with thyroid disease. PATIENTS AND METHODS TG, TPO, and RET/PTC1 mRNA were analyzed in 52 peripheral-blood samples from 44 patients diagnosed with thyroid carcinoma (24 patients), adenoma (five patients), and nodular hyperplasia (15 patients) by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS TG and TPO were identified in 13 patients (54.2%) with thyroid carcinoma, which includes five of eight patients with no clinical evidence of disease at the time of blood collection. Four of 5 patients had cervical lymph node metastases and/or extrathyroid extension at the time of the initial surgery. RET/PTC1 mRNA was detected in the peripheral blood of only one patient with papillary thyroid carcinoma. This sample was also positive for TG and TPO. TG and TPO were detected in two patients (10%) with benign thyroid nodules. All positive samples from patients with benign thyroid lesions were collected before surgery, whereas all samples collected after surgery were negative. RET/PTC1 mRNA was not detected in any of the patients with benign thyroid nodules. RT-PCR positivity for TG and TPO mRNA was higher in patients with carcinoma than in patients with benign lesions (P = .002). CONCLUSION TG, TPO, and RET/PTC1 mRNA are detectable in the peripheral blood of patients with thyroid disease, which correlates with a diagnosis of carcinoma.

Highly Sensitive Nonradioactive Single-strand Conformational Polymorphism: Detection of Ki- ras Mutations

Mutation detection by single-strand conformational polymorphism (SSCP) analysis is more difficult when the variant is limited to a small proportion of target sequences in a sample. Use of SYBR-Green II, a sensitive, nonradio-active, minimally hazardous nucleic acid stain, permits detection of Ki-ras mutants present as less than 0.5% of the target sequences. The polymerase chain reaction (PCR) primers we have selected produce an amplicon that distinguishes all clinically observed variants in Ki-ras codons 12 and 13 from the wild type. We compared mutant discrimination and SYBR-Green II detection sensitivity in three formats: (a) standard MDE gel SSCP, (b) rapid minigel MDE using an internal gel temperature controller, and (c) rapid resolution in chilled 15% (37.5:1) acrylamide minigels. All these gels are easily evaluated by standard ultraviolet transillumination and digital image analysis. This ssDNA staining method is rapid, highly reproducible, and minimally hazardous, and minigels use 25% the reagents of most other systems. Our improvements are relevant for the detection of mutations in pathologic samples with minimal targets, such as fine-needle aspirates, and body fluids in which mutated alleles of a gene may be present at low levels but carry a high level of diagnostic or prognostic importance.

Ki-ras mutations and the carcinoembryonic antigen level in fine needle aspirates of the pancreas

OBJECTIVE To evaluate the sensitivity and specificity of the carcinoembryonic antigen (CEA) immunoassay and Ki-ras genotyping as adjuncts to the cytologic diagnosis of pancreatic fine needle aspirates (FNAs). STUDY DESIGN A retrospective study of 30 patients with pancreatic masses evaluated with CEA immunoassay and gel or hybridization analysis of allele-specific polymerase chain reaction for mutant Ki-ras (codons 12 and 13). DNA was isolated from fixed, paraffin-embedded samples. Diagnoses were correlated with cytologic evaluations and patient outcome. RESULTS Diagnoses included 17 pancreatic carcinomas, 3 other malignancies and 10 benign lesions. Sixty-five percent of all FNAs had mutated Ki-ras, and 42% of samples with altered Ki-ras had multiple mutations. Replicate FNA samplings in five of six patients had concordant genotypes. Sensitivities for diagnosis were as follows: cytology alone, 76%; CEA alone, 82%; Ki-ras alone, 82%; cytology plus CEA, 100%; cytology plus Ki-ras, 94%. Although specificities for Ki-ras (30%) and CEA (50%) individually were low, elevated CEA level and mutated Ki-ras in a sample with negative cytology strongly indicated false negative cytology. CONCLUSION The addition of either or both the CEA assay and Ki-ras mutation analysis enhances the sensitivity of the cytologic diagnosis of pancreatic carcinoma by FNA.

Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells.

We have studied the mechanism of cellular resistance to cardiac glycosides in C+ cells. C+ cells were resistant to ouabain and overproduced plasma membrane-bound Na,K-ATPase relative to parental HeLa cells. Overexpression of Na,K-ATPase in C+ cells correlated with increased ATPase mRNA levels and amplification (approximately 100 times) of the ATPase gene. Growth of C+ cells in ouabain-free medium resulted in a marked decline in ATPase mRNA and DNA levels. However, when cells were reexposed to ouabain, they proliferated and ATPase mRNA and DNA sequences were reamplified. Restriction analysis of C+ and other human DNA samples revealed the occurrence of rearrangements in the region of the Na,K-ATPase gene in C+ cells. Furthermore, C+ cells expressed an ATPase mRNA species not found in HeLa cells. These results suggest that amplification of the gene coding for Na,K-ATPase results in overproduction of Na,K-ATPase polypeptides. Amplification of the ATPase gene or the expression of new ATPase mRNA sequences or both may also be responsible for acquisition of the ouabain-resistant phenotype.

Insulin induction of tyrosine aminotransferase in synchronized hepatoma cells

Abstract Addition of insulin to unsynchronized rat hepatoma cells, previously incubated with dexamethasone, promotes a rapid increase in tyrosine aminotransferase activity. Mitotic cells in the presence or absence of colcemid are not inducible by either insulin or dexamethasone. Inducibility by dexamethasone is restored only after the third hour of the G1 phase, presumably related to the appearance of the putative posttranscriptional repressor ( Martin, D.W. et al. (1969). Proc. Nat. Acad. Sci., 63 : 842–849 ). In contrast, inducibility by insulin is restored immediately upon passage of the synchronized cells into G1. These data suggest that insulin can induce tyrosine aminotransferase in the absence of the repressor.

The Molecular Biology of the Na,K-ATPase and Other Genes Involved in the Ouabain-Resistant Phenotype

Na,K-ATPase is the membrane-embedded enzyme responsible for the active transport of sodium and potassium ions in most animal cells. This protein, commonly referred to as the sodium pump, plays a central role in mediating the electrical activity of the brain. In neurons, the activity of the sodium pump maintains the ion gradients that provide the driving force for the action potential (Thomas, 1972). The uptake of neurotransmitters and the efflux of calcium from neurons are also coupled to the activity of the ATPase (Iverson and Kelly, 1975). In glia, the sodium pump plays a critical role in potassium uptake during periods of intense neuronal activity (Hertz, 1977).