Assaying DNA methylation based on high-throughput melting curve approaches

Here we describe two high-throughput methods to assay DNA methylation, melting curve methylation specific PCR (McMSP) and melting curve combined bisulfite restriction analysis (McCOBRA), which adapt standard MSP and COBRA methods to a melting curve analysis based platform. We show that McMSP and McCOBRA can accurately determine methylation status in a high-throughput and gel-free manner. Moreover, McCOBRA can be used to quantitatively estimate the percent of methylated DNA at a specific CpG site within a heterogeneous sample. The accuracy of McMSP and McCOBRA was initially tested using the 5'-CpG site of the tumor-suppressor gene CDKN2A as a model system in homogeneous and heterogeneous controls, and cancer cell line samples. Furthermore, the robustness of McMSP and McCOBRA was validated in four additional loci. We demonstrate that McCOBRA and McMSP provide several advantages over existing methods, as they are simple, accurate, and high-throughput, which makes them widely applicable to large-scale methylation studies.     

Integral method for measuring the quantity of cellular DNA content by digital microphotography

Quantitative measurements of nuclear DNA content based on Feulgen reaction and the analysis of CCD images has been proposed. The measurements were performed in the monochrome CCD option (650 × 514 pixels) with a wavelength of 551 nm. The linear dependence of photomatrix element signals on the falling light was shown with a multigrade light absorption filter. The optimal microscope and camera settings and an approach for elimination of the optic blur are proposed. It was found that the contribution of background fluorescence of Feulgen-stained nuclei into the measurements was negligible. Densitometric measurements of the DNA content in blood cells of four vertebrate species (Gallus domesticus, Danio rerio, Homo sapiens, Rana arvalis) were consistent with the literature data. The precision of our approach is comparable to other known cytometry methods (). The current improvement of CCD technical parameters and the widespread use of CCD cameras in biological applications give perspectives for the development of the suggested approach for measuring the quantity of cellular DNA.     

Determination of melting temperature and temperature melting range for DNA with multi-peak differential melting curves

Many factors that change the temperature position and interval of the DNA helix–coil transition often also alter the shape of multi-peak differential melting curves (DMCs). For DNAs with a multi-peak DMC, there is no agreement on the most useful definition for the melting temperature, T_m, and temperature melting width, ΔT, of the entire DNA transition. Changes in T_m and ΔT can reflect unstable variation of the shape of the DMC as well as alterations in DNA thermal stability and heterogeneity. Here, experiments and computer modeling for DNA multi-peak DMCs varying under different factors allowed testing of several methods of defining T_m and ΔT. Indeed, some of the methods give unreasonable “jagged” T_m and ΔT dependences on varying relative concentration of DNA chemical modifications (r_b), [Na⁺], and GC content. At the same time, T_m determined as the helix–coil transition average temperature, and ΔT, which is proportional to the average absolute temperature deviation from this temperature, are suitable to characterize multi-peak DMCs. They give smoothly varying theoretical and experimental dependences of T_m and ΔT on r_b, [Na⁺], and GC content. For multi-peak DMCs, T_m value determined in this way is the closest to the thermodynamic melting temperature (the helix–coil transition enthalpy/entropy ratio).     

Rapid competitive PCR using melting curve analysis for DNA quantification.

A rapid competitive PCR method was developed to quantify DNA on the LightCycler. It rests on the quantitative information contained in the melting curves obtained after amplification in the presence of SYBR Green I. Specific hybridization probes are not required. Heterologous internal standards sharing the same primer binding sites and having different melting temperatures to the natural PCR products were used as competitors. After a co-amplification of known amounts of the competitor with a DNA-containing sample, the target DNA can be quantified from the ratio of the melting peak areas of competitor and target products. The method was developed using 16S rDNA fragments from Streptococcus mutans and E. coli and tested against existing PCR-based DNA quantification procedures. While kinetic analysis of real-time PCR is well established for the quantification of pure nucleic acids, competitive PCR on the LightCycler based on an internal standardization was found to represent a rapid and sensitive alternative DNA quantification method for analysis of complex biological samples that may contain PCR inhibitors.     

DNA force-extension curve under uniaxial stretching

Single-molecule experiments indicate that a double-stranded DNA (ds‐DNA) increases in length if put under tension greater than 10 pN; beyond this point, its conformation can no longer be described using an inextensible worm-like chain model. For this purpose, a general sequence-dependent elastic model for tensions greater than 10 pN and for both single-stranded (ss) and ds‐DNA is proposed, and the effective elastic bending and torsional rigidities are determined from experiments to characterise their deformation. The key to this progress is that the bending and torsional deformations of the DNA backbones, the base-stacking interactions and the hydrogen bond force between the complementary base pairs are quantitatively considered in this model. Moreover, this simple elastic model can be used to globally fit to the abrupt B–S experimental transition data over a wide range of DNA molecule extensions. Based on this robust model, further study may be warranted on the mechanical response of ss- and ds‐DNA molecules.     

Determination of the A-T content of DNA by second-derivative ultraviolet spectroscopy

A method for the determination of the A-T content of DNA based on second-derivative ultraviolet spectra is presented. It allows measurement in a wide range of pH values, ionic strengths, and buffer media. It is nondestructive for the sample and requires not more than 10 micrograms of DNA.     

Determination of the DNA content of human chromosomes by flow cytometry

The mean relative DNA content of each human chromosome was calculated from flow karyotypes of ethidium bromide-stained chromosomes obtained from healthy, normal individuals. These values were found to correlate closely with previously published data obtained by photometric scanning of stained, fixed chromosomes. Calculations of the normal variation in DNA content of each human chromosome indicated that chromosomes 1, 9, 16, and Y (chromosomes with large centric heterochromatic regions) were the most variable, followed by the acrocentrics, 13, 14, 15, 21, and 22. Chromosomes 2, 3, 18, and 19 were also found to vary significantly in DNA content. Chromosomes from a number of subjects with extreme heteromorphisms were flow karyotyped to obtain an estimate of the extent of variation in DNA content of each chromosome. The greatest difference between extreme variants was found for chromosome 1 (which differed by 0.82% of the total genomic DNA), followed by 16 and 9. The largest Y-chromosome variant was 85.9% bigger than the smallest. The precise karyotype analysis produced by flow cytometry resolved many differences between chromosome homologs, including some that cannot be readily distinguished cytogenetically. The implications of these findings for detection of chromosome abnormalities by flow karyotype analysis are discussed.     

Empirical likelihood inference for the area under the ROC curve

For a continuous-scale diagnostic test, the most commonly used summary index of the receiver operating characteristic curve (ROC) is the area under the curve (AUC) that measures the accuracy of the diagnostic test. In this article, we propose an empirical likelihood (EL) approach for the inference on the AUC. First we define an EL ratio for the AUC and show that its limiting distribution is a scaled chi-square distribution. We then obtain an EL-based confidence interval for the AUC using the scaled chi-square distribution. This EL inference for the AUC can be extended to stratified samples, and the resulting limiting distribution is a weighted sum of independent chi-square distributions. Additionally we conduct simulation studies to compare the relative performance of the proposed EL-based interval with the existing normal approximation-based intervals and bootstrap intervals for the AUC.     

Determination of pyrimidine nucleotide sequences of DNA]

A modification of the Burton method for determination of pyrimidine nucleotide blocks (isopliths) of DNA, providing a higher yield of large-sized nucleotide isopliths, is described. The amount of side products (interisopliths) does not exceed their amount upon DNA hydrolysis according to the Burton method. Another advantage of the technique recommended is a considerable shortening of hudrolysis time (20 min instead of 18 hours). The modification described has been successfully used to determine the pyrimidine nucleotide blocks of some warm-blooded animals DNAs. It has been found that the DNA of animals with higher sensitivity to ionised irradiation contains more oligothymidylic sequences as compared to the DNA of animals, less sensitive to irradiation.     

Determination of DNA content of aquatic bacteria by flow cytometry

The distribution of DNA among bacterioplankton and bacterial isolates was determined by flow cytometry of DAPI (4',6'-diamidino-2-phenylindole)-stained organisms. Conditions were optimized to minimize error from nonspecific staining, AT bias, DNA packing, changes in ionic strength, and differences in cell permeability. The sensitivity was sufficient to characterize the small 1- to 2-Mb-genome organisms in freshwater and seawater, as well as low-DNA cells ("dims"). The dims could be formed from laboratory cultivars; their apparent DNA content was 0.1 Mb and similar to that of many particles in seawater. Preservation with formaldehyde stabilized samples until analysis. Further permeabilization with Triton X-100 facilitated the penetration of stain into stain-resistant lithotrophs. The amount of DNA per cell determined by flow cytometry agreed with mean values obtained from spectrophotometric analyses of cultures. Correction for the DNA AT bias of the stain was made for bacterial isolates with known G+C contents. The number of chromosome copies per cell was determined with pure cultures, which allowed growth rate analyses based on cell cycle theory. The chromosome ratio was empirically related to the rate of growth, and the rate of growth was related to nutrient concentration through specific affinity theory to obtain a probe for nutrient kinetics. The chromosome size of a Marinobacter arcticus isolate was determined to be 3.0 Mb by this method. In a typical seawater sample the distribution of bacterial DNA revealed two major populations based on DNA content that were not necessarily similar to populations determined by using other stains or protocols. A mean value of 2.5 fg of DNA cell(-1) was obtained for a typical seawater sample, and 90% of the population contained more than 1.1 fg of DNA cell(-1).     

Determination of the 3'' terminal nucleotide of DNA fragments

A new method for determining the 3'-terminal nucleotide of a DNA strand is presented. Use is made of the fact that one (and only one) 2',3'-dideoxyribonucleotide can be added to the 3'-end of a DNA fragment with calf thymus terminal transferase. Addition of more than one nucleotide analog per strand is impossible due to the absence of a 3'-terminal hydroxyl group. If the terminatind dideoxyribonucleotide contains an (alpha 32p) label, the resulting 3'-blocked strand can be digested by "nearest neighbor" techniques and the original 3'-endgroup determined. Picomole quantities of DNA strands can be labeled and the 3'-end determined.     

Characteristics of nucleotide blocks in coding and non-coding DNA sequences from different organisms

A statistical analysis of occurrence of particular nucleotide runs (1 divided by 10 nucleotides long) in DNA sequences of different species has been carried out. There are considerable differences in run distributions in DNA sequences of prokaryotes, invertebrates and vertebrates. Distribution of various types of runs has been found to be different in coding and non-coding sequences. There is an abundance of short runs 1 divided by 2 nucleotides long in coding sequences, and there is a deficiency of such runs in the non-coding regions. However, some interesting exceptions from this rule exist: for run distribution of adenine in prokaryotes and for distribution of purine-pyrimidine runs in eukaryotes. This may be stipulated by the fact that the distribution of runs are predetermined by structural peculiarities of the entire DNA molecule. Runs of guanine or cytosine of three to six nucleotides long occur predominantly in the non-coding DNA regions in eukaryotes, especially in vertebrates.     

Determination of guanine-plus-cytosine content of bacterial DNA by dual-laser flow cytometry

A dual-laser flow cytometer was used to analyse different species of bacteria for the molar percentage of guanine-plus-cytosine (% G + C) without the need for DNA extraction or purification. Ethanol-fixed bacterial cells were stained with a combination of DNA-specific fluorochromes, Hoechst 33258 and chromomycin A3, which bind to AT- and GC-rich regions of DNA, respectively. A linear relationship (r = 0.99) was demonstrated between the log of the ratio of chromomycin A3 to Hoechst 33258 fluorescence and the log of the % G + C as determined by thermal denaturation (Tm) or buoyant density centrifugation (Bd) methods. Linearity was maintained for all bacterial species tested over the range of 28-67% G + C. A standard curve was constructed using five strains whose % G + C had been determined by other methods. From the equation describing this line, the % G + C values of nine other strains with known DNA base composition, together with the five strains used to construct the curve, were calculated using the chromomycin A3 to Hoechst 33258 ratio and were in agreement with values obtained by Tm, Bd or HPLC. The reproducibility of flow cytometric analysis (mean error 0.7% G + C) compared well with the reproducibility of other methods. Mixtures containing two species were also analysed. Two cell populations could be discerned in mixtures containing two species which differed in base composition by as little as 4% G + C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of a nucleotide sequence in bacteriophage f1 DNA by primed synthesis with DNA polymerase

A method is described for the determination of nucleotide sequences in DNA by using specific oligonucleotides as primers for copying specific regions by DNA polymerase. The method was applied to bacteriophage f1 DNA using the synthetic octanucleotide A-C-C-A-T-C-C-A as primer and a sequence (sequence A) of 81 nueleotides was determined. Synthesis was carried out in the presence of manganese and with one of the deoxyribotriphosphates (dCTP or dGTP) replaced by the corresponding ribotriphosphate so that mixed oligonucleotides were found which could be specifically split at the ribonucleotide residues by the appropriate ribonuclease or by alkali. The relative order of the digestion products was determined by fractionating the undigested oligonucleotides according to size on a two-dimensional system and digesting the isolated products. In the presence of rGTP the octanucleotide appeared to prime at a second site giving rise to a second sequence (B) besides sequence A. The complementary sequence to sequence A, which corresponds to the plus strand of f1 DNA and to the messenger RNA, contains five nonsense codons, four of which are in the same phase, and two possible initiation codons. It also contains a repetitive sequence which suggests its evolutionary origin by duplication.