EP1402072A4 - ACCURATE AND EFFICIENT QUANTIFICATION OF REAL-TIME PCR DNA SENSITIVITY - Google Patents

ACCURATE AND EFFICIENT QUANTIFICATION OF REAL-TIME PCR DNA SENSITIVITY

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Publication number
EP1402072A4
EP1402072A4 EP02752010A EP02752010A EP1402072A4 EP 1402072 A4 EP1402072 A4 EP 1402072A4 EP 02752010 A EP02752010 A EP 02752010A EP 02752010 A EP02752010 A EP 02752010A EP 1402072 A4 EP1402072 A4 EP 1402072A4
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Prior art keywords
dna
modifying agent
chromatin
genome
dnasel
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German (de)
English (en)
French (fr)
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EP1402072A1 (en
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John A Stamatoyannopoulos
Michael O Dorschner
Michael Mcarthur
Georges Stamatoyannopoulous
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • the invention relates generally to methods of DNA analysis and more specifically to methods for analysis of genomic sequences having desirable biochemical activity.
  • Quantification of the signal between this sub-band and the parental band can be compromised by several variables: there may be a different transfer efficiency in blotting the parental and smaller-sub band; disperse cutting will form a smeary sub- band; the efficiency of hybridization of the probe to the two bands may differ; analysis may become confused if more than one hypersensitive site exists on the same parental band.
  • Southern hybridization assay Another problem with Southern hybridization assay is that this generally allows only analysis of relatively large segments of material (the size of the parental band) and mapping of the position of novel DNasel-hypersensitive site depends upon the resolution of the gel.
  • the PCR approach allows sequences to be amplified which can be tested directly as to whether or not they are hypersensitive to digestion. Also the distribution of cutting can be established by designing primer pairs amplifying adjacent, or overlapping, sequences. Structural information can be gathered for the first time about the status of sequences proximal to the hypersensitive sites and the effect of their presence to the local chromatin structure.
  • the present invention overcomes the problems and disadvantages associated with current strategies and designs and provides methods for accurately and efficiently analyzing a region of a genome.
  • An embodiment of the invention is a method for accurately and efficiently determining sensitivity of a candidate region of a genome to a DNA modifying agent comprising isolating chromatin from a population of eukaryotic cells containing said genome; treating at least one portion of said isolated chromatin with said DNA modifying agent under conditions to cause DNA strand breakage; treating another portion with said DNA modifying agent under modified conditions; isolating treated DNA from the portions; amplifying the candidate region from isolated DNA by realtime PCR from each portion by real time PCR with a set of primers and obtaining a signal; determining a relative copy number of said candidate region within each isolated DNA portion by: determining the copy number of said candidate region in each of a plurality of DNA samples each containing a different amount of DNA in a fixed proportion with respect to each other and thereby calculating a first standard copy number curve; determining the copy number of a reference region in each of a plurality of DNA samples each containing a different amount of DNA in a fixed proportion with respect to each other and
  • the genome is a human genome.
  • the candidate sequence is less than 250 base pairs in length.
  • the candidate region is between about 50 and about 2,000 base pairs in length, hi yet another embodiment one portion of isolated chromatin comprises from 0.15 pg to about 5 ug of nucleic acid.
  • the conditions and the modified conditions are selected from the group consisting of different concentrations of the DNA modifying agent (e.g. pM, nM, mM), different times (e.g. seconds, minutes, hours), different temperatures (e.g. 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C), different buffer conditions (e.g. high to low pH, ion concentration or buffering capacity), different concentrations of chromatin (e.g. one copy of genome to 5 ug), and combinations thereof.
  • the conditions and the modified conditions are performed at the same temperature (e.g.
  • amplifying each isolated DNA by the real-time PCR proceeds through at least eighteen cycles. In another embodiment amplifying each isolated DNA by the real-time PCR proceeds through at least thirty-five cycles.
  • the set of primers amplify fragments of DNA that are from about 200 to 400 base pairs in length.
  • the reference region is insensitive to the DNA modifying agent. In another embodiment the reference region is more sensitive to the DNA modifying agent. In another embodiment the reference region is less sensitive to the DNA modifying agent. In another embodiment the reference sequence is less than about 250 base pairs in length. In another embodiment the reference region is between about 50 and about 2,000 base pairs in length.
  • the reference region is between 200 and 400 base pairs in length, h another embodiment a double strand DNA specific marker is used during PCR to detect the accumulation of PCR products as a function of the number of rounds of amplification.
  • the DNA modifying agent is DNase I and the DNA modifying reactions utilize a single reaction time point and differing concentrations of DNase I.
  • the DNA modifying agent introduces single stranded nicks into the DNA.
  • the sensitivity of a genome of a eukaryotic species comprising: combining cells of a first eukaryotic species with a first genome with cells from a second eukaryotic species with a second genome having a combined cell population totaling at least about 10 8 cells, about 10 9 cells, or about 10 10 cells; isolating chromatin from the combined cell population and dividing the isolated chromatin into one or more portions; treating one portion or sub-portion of chromatin with a first amount of said DNA modifying agent; isolating treated DNA from the portions; amplifying each isolated DNA by real time PCR with a set of primers that amplify a template sequence of the first eukaryotic species; and determining the sensitivity of said template sequence to the DNA modifying agent within the first genome.
  • the cells of the first eukaryotic species number less than 10 7 .
  • the cells of the first eukaryotic species comprise less than about 10 6 cells.
  • the DNA modifying agent introduces single stranded nicks into the DNA.
  • Figure 2 shows the DNasel-sensitivity of NF-M and HS2 in mouse fetal liver. Chromatograms were generated on the Lightcycler system (Roche) following Real-time quantitative PCR experiments performed on a series of genomic DNAs isolated from DNasel-digested fetal liver nuclei amplifying (a), Nf-M and (b), HS2. Using the LightCycler FastStart DNA Master SYBR green I mix at lx final concentration (containing PCR buffer, dNTPs, MgCl 2 and Taq polymerase) the QCPR reactions were assembled as follows: 0.3uM of each primer, additional MgCl 2 to 3mM and 5-30ng of template DNA.
  • Reactions are thermal-cycled under two sets of conditions depending on amplicon size; 250 bp or 500 bp.
  • 250 bp amplicons the following amplification protocol is used: 95°C for 10 minutes, followed by 40 cycles of 95°C; 5 sec, 60°C; 5 sec and 72°C; 15 sec.
  • 500 bp amplicons the 72°C phase is extended to 25 sec. Both amplification protocols were followed by a melting curve analysis.
  • Each panel shows the progress of the PCR reactions plotted as a gain in fluorescence (on a logarithmic scale), due to the binding of the dsDNA-specific SYBR-green dye to the PCR product, as a function of the number of. cycles.
  • Figure 3c The standard curves in Figure 3c were used to calculate the percentage of copies of the HS2 amplicon remaining in 50 ng of DNasel-treated genomic DNA. The amount of template DNA was standardized by correcting for amplification of the DNasel- insensitive Nf-M sequence.
  • Figure 5 displays plots of the DNasel-digestion profiles of amplicons throughout the mouse ⁇ -globin LCR. The plots were generated as described in the legend to Figure 4. The primer pairs used were those shown in Figure 1; (a), Nf-M and the DNasel-hypersensitive sites HSI to HS4 and (b), the Flanking sequences
  • Figure 6 displays a plot of all the DNasel-digestion profiles generated in this study. Four distinct classes of profiles are evident and are labeled as described in the text.
  • Figure 7 displays a schematic diagram to account for how DNasel probes accessibility. The consequence of competition between DNasel (shown as a solid circle) and a nucleosome (a hollow oval) for the extent of restriction of a molecule of duplex DNA in the case of (a), naked DNA, (b), partially bound and (c) covered DNA.
  • the present invention is directed to methods for accurately and efficiently analyzing a region of a genome.
  • Embodiments of the invention include several desirable features that further alleviate problems in the field as cited above.
  • a first particularly desirable embodiment is a method for analyzing chromatin structure from limiting amounts of tissue. This embodiment allows the use of as little as 10 5 cells for an analysis. The cells of interest can be mixed in a larger population of cells from a different species. This material can be used to generate the DNasel-digestion series of genomic DNAs used in the analysis. It is preferred to use up to 10 8 cells in each DNasel-digestion.
  • a second particularly desirable embodiment allows the detection of single- stranded nicks that have been introduced into chromatin sequences.
  • the method need not just be applied to genomic DNAs isolated from nuclei treated with DNasel but can use any DNA-modifying agent.
  • Southern hybridization fails to detect single-stranded nicks introduced (as the DNA molecules migrate as duplexes) the PCR-based assay detects the cut as it destroys one half of the template.
  • Several sensitive DNA modifying agents, such as hydrogen peroxide which has the advantage over DNasel of having no sequence specificity of cutting and being entirely soluble, allows much higher resolution in its cutting pattern and introduces single stranded nicks only.
  • DNA cutting agents and methods for cutting DNA are available and specifically contemplated for embodiments of the invention.
  • epigenetic modifications in chromatin such as histone acetylation and cytosine methylation may be used.
  • Further optional treatments include contact with one or more of the following DNA-modifying agents or conditions: nucleases (both sequence-specific and non-specific); topoisomerases; methylases; acetylases; chemicals; pharmaceuticals (e.g., chemotherapy agents); radiation; physical shearing; nutrient deprivation (e.g., folate deprivation), and other agents that are commercially available and known to those of ordinary skill.
  • proteins and RNAs which control the structure of the nucleus are being identified, and these also could be used as targets for modifying DNA.
  • proteins that bind to a given DNA sequence or set of sequences may be employed to induce DNA modification such as strand breakage. Proteins can either be modified by many means, such as incorporation of 125 I, the radioactive decay of which would cause strand breakage (e.g., Acta Oncol. 39: 681-685 (2000)), or modifying cross- linking reagents such as 4-azidophenacylbromide (e.g., Proc. Natl. Acad. Sci. USA 89: 10287-10291) which form a cross-link with DNA on exposure to UV-light. Such protein-DNA cross-links can subsequently be converted to a double-stranded DNA break by treatment with piperidine.
  • DNA modification relies on antibodies raised against specific proteins bound at one or more DNA sites, such as transcription factors or architectural chromatin proteins, and used to isolate the DNA from nucleoprotein complexes.
  • An example of a currently used technique cross-links proteins and DNA within the eukaryotic genome following treatment with formaldehyde, for example, and after isolation of the chromatin and following either sonication or digestion with nucleases the sequences of interest are immunoprecipitated (Orlando et al. Methods 11: 205-214, 1997).
  • Another modification is cytosine methylation. The global pattern of methylation is relatively stable but certain genetic control regions become methylated if they are silenced or conversely demethylated if activated.
  • Differential methylation can be detected by use of pairs of restriction endonucleases that cut the same site differently according to whether or not it is methylated (Tompa et al. Curr. Biol. 12: 65-68, 2002).
  • genomic sequencing a methodology developed by Pfeifer et al. Science 246: 810-813, 1989
  • This material can be used as a template in PCR with primers sensitive to the C to U transition.
  • the potential mismatch (G:U) between oligonucleotide and template can be cleaved by E.coli Mismatch
  • a further approach is directed to the enzymatic machinery which gives rise to or maintains the epigenetic patterns.
  • This machinery can also be labeled as described above so that it can be induced to cause detectable DNA modifications such as double stranded DNA breaks.
  • Target proteins for this kind of approach would include the recently described HATs (Histone-Acetyl Transferases), HDACs (Distone De-Acetylase Complexes) whose effect on transcriptional induction has been recently described (Cell 108: 475-487, 2002), as well as DNA methyltransferases and structural proteins that bind to the sites of methylation, such as MeCPl and MeCP2. Histones, and transcription factors, are also known to become methylated, phosphorylated and ubiquitylated.
  • nuclei By contrast specific areas of eukaryotic nuclei have been shown to be transcriptionally inert (Nature 381: 529-531, 1996) and associated with heterochromatin. Fractionation of the nucleus on the basis of such and similar physical properties can be used to selectively cleave DNA.
  • a third particularly desirable embodiment allows the quantitative analysis of naturally occurring single-stranded DNA structures in vivo.
  • the presence of single- stranded DNA in nuclei is unusual and can be caused by the action of enzymes, such as topoisomerase I, as a transient consequence of DNA replication or from formation of unusual DNA structures (such as Z-DNA or triplex DNA).
  • enzymes such as topoisomerase I
  • the kinetics of digestion of single strand-specific cutters, such as potassium permanganate, or primer-directed restriction can be effectively monitored using the assay described above.
  • the formation of triplex DNA structures is of particular interest as these are believed to be involved in regulation of downstream genes and it would be of interest to establish if there were a correlation between their formation and expression.
  • Real-time PCR was used to allow quantification of the sensitivity of chromatin to digestion by DNasel.
  • This approach has three clear advantages to the more conventional use of the Southern hybridization assay: the accuracy of quantification is improved; the resolution of the assay is enhanced- by designing primers to amplify small amplicons so that it is possible to analyze sequences both eo-incident and proximal to sites of DNasel-hypersensitivity; less material is needed, as little as 5 ng of treated genomic DNA.
  • This method was applied in an analysis of the chromatin structure of the previously described mouse ⁇ -globin locus control region (LCR) using fetal liver cells.
  • LCR mouse ⁇ -globin locus control region
  • the four hypersensitive sites of the canonical mouse LCR, HSI to HS4, are shown to have kinetics of digestion consistent with these sequences being nucleosome-free in vivo.
  • a different pattern was seen for HS6 (a recently described 'weak' hypersensitive site). The site was also rapidly lost but more of the sites proved resistant, consistent with only a portion of HS6 being nucleosome-free. This finding implies that in vivo the LCR is structurally heterogeneous. Sequences proximal to the hypersensitive sites show a third pattern of intermediate sensitivity, consistent with the chromatin being unfolded but the sites still bound by a continual nucleosomal array.
  • LCRs locus control regions
  • HSI tissue-specific DNasel- hypersensitive sites
  • Primers are designed to separately amplify similar-sized products from either the LCR or a known DNasel-insensitive gene, Nf-M (which is used as an internal control for the amount of template), from a series of genomic DNAs isolated from DNasel-treated nuclei. By reference to a standard curve, it is possible to calculate the number of amplicons destroyed as a function of DNasel concentration. These data allow an examination of the kinetics of digestion for each site and an accurate determination of the proportion of sites which proved DNasel-insensitive. This approach was used to investigate the chromatin structure of the murine beta-globin locus as an exemplary embodiment.
  • Mouse fetal liver was DNasel-digested in samples by harvesting twenty fetal livers from 11.5- 12.5 d.p.c mouse embryos. The livers were pooled and dispersed in a loose fitting homogenizer in 5 ml buffer A (15 mM TrisHCl pH 7.6, 60 mM
  • the samples were treated with Proteinase K overnight and DNA recovered after phenol-chloroform extraction and ethanol precipitation.
  • the DNA was then dialysed against two changes of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA), re-precipitated and diluted in water to a concentration of 50 ng/ ⁇ l as measured by DNA flurometry.
  • DNasel-sensitivity studies were carried out by performing real-time quantitative PCR on the DNA samples using the Roche Molecular-Biochemical's Lightcycler system. PCR reactions were performed using the manufacturer's SYBR- green master kit optimized for use with the following primer pairs (see Figure 1 for the positions of the amplicons within the ⁇ -globin LCR; the co-ordinates of the sequence of the amplified fragment and the GI accession number of the sequence file are shown): HSIf, 5'-AGA TTA TAT TGC CAT GGT ACA CTT GAA-3' (SEQ ID NO 1);
  • Fklr 5'-CAT TGG CAG AAA GCT CTC ATA CA-3' (SEQ ID NO 4), (5709-6179; GI: 50150);
  • Fk2f 5'-GGA TTT TAC TAT ATA ACT ATG CTA TCA-3' (SEQ ID NO 5); Fk2r 5'AGA AAG TAA GGG ATGACG TGT AAT ACA AC-3' (SEQ ID NO 6),
  • HS2r 5'-CAC ACA GCA AGG CAG GGT C-3' (SEQ ID NO 8), (11767-12237; GI: 50150);
  • Nf-Mr 5'-GCG GCA TTT GAA CCA CTC TT-3' (SEQ ID NO 18), (988-1438; GI: 53357).
  • PCR was performed using FastStart DNA SYBR-green kit (Roche) as per the manufacturer's instructions. The reactions were performed in a volume of 10 ⁇ l with 0.5, 5 and 50 ng of genomic DNAs isolated froze the mouse erythroleukemia line (MEL) in order to prepare standard curves for each of the amplicons, or 50 ng of sample from each DNasel-digestion point. The number of copies of target remaining intact, corrected for the size of the fragment, was calculated by reference to the standard curve. These data were then plotted versus the units of DNasel used to digest the nuclei.
  • MEL mouse erythroleukemia line
  • An alternative protocol is to use the LightCycler FastStart DNA Master SYBR green I mix at lx final concentration (containing PCR buffer, dNTPs, MgCl 2 and Taq polymerase), and to assemble the QPCR reactions as follows: 0.3 uM of each primer, additional MgCl to 3 mM and 5-30 ng of template DNA. Reactions are thermal-cycled under two sets of conditions depending on amplicon size; 250 bp or
  • 500 bp For 250 bp amplicons the following amplification protocol is used: 95°C for 10 minutes, followed by 40 cycles of 95°C; 5 sec, 60°C; 5 sec and 72°C; 15 sec. For 500 bp amplicons the 72°C phase is extended to 25 sec. Both amplification protocols are followed by a melting curve analysis. Quantification of DNasel-digestion by Real-time PCR
  • genomic DNA samples that were tested had been harvested from mouse fetal live nuclei that had been treated with increasing amounts of DNasel, in order to establish the sensitivity of their chromatin structure to digestion. Typically 50 ng of genomic DNA was used for each reaction but it was possible to use as little as 5 ng. A serial dilution of undigested genomic DNA was also analyzed (using 0.5 ng of material as the earliest point) in order to produce a standard curve so that the number of copies of template in each of the samples could be calculated. In order to correct for small differences in the amount of DNA the separate amplification of a similarly- sized fragments from a known DNasel-insensitive gene, Nf-M (22), was used as an internal control for the amount of DNA present in the reaction. A Southern hybridization assay confirmed that there was no detectable digestion of this gene under the conditions used (data not shown). Chromatograms from the Roche Molecular Biochemical's Lightcycler
  • This number of cycles, the C T value is calculated by extrapolation from the linear part of the curve (identified by the two gray crosses) where amplification is exponential and plots the intercept with the threshold line, represented by the red crosses.
  • the data used to generate the standard curve for each experiment show a regular increases in the Cj value as the amount of template is decreased by a factor to ten.
  • a plot of the log of concentration of template against C T value generates a straight line which is used to calculate the percentage of remaining amplicons in the DNasel-digested samples (Figure 3c).
  • the digestion profile can be seen by plotting the percentage of copies of HS2 remaining, corrected for DNA content, against the number of units of DNasel used in the digestion of nuclei ( Figure 4). Two general features of the curve are apparent; the initial rate of loss of copies is very fast and the curve reaches a plateau, representing the number of sites that are not accessible to digestion. The first property is an indication of the level of sensitivity of the site to digestion (a less accessible site would be expected to digest more slowly, leading to a slower rate). The second establishes the proportion of material which remains inaccessible, due to the cells being derived from the non-erytl roid compartment of the tissue where the hypersensitive site does not form.
  • the profiles of the intervening sequences are similar to each other and are plotted in a shaded compartment of Figure 5b. They show a less steep rate of loss of copy number than with the hypersensitive sites and the curve plateaus later and a higher level.
  • the final profile recorded is that of the 'weak' DNasel- hypersensitive HS6 ( Figure 5b); the rate of loss is similar to that of the other hypersensitive sites and it reaches a plateau early, but it is at a higher level. All four types of profile are plotted together in Figure 6.
  • a fast rate of digestion is consistent with the site being extremely accessible in the nuclei to digestion, as it is expected to occur at a hypersensitive site where the nucleosomal array is interrupted allowing the DNasel enzyme free access to cut.
  • the enzyme can independently cut twice to completely destroy the site.
  • a slower rate of digestion is presumably caused by the site being blocked in vivo, most likely by a nucleosome, and this competing with DNasel for access to the site ( Figure 7b). If cutting does occur here it may be more likely to be a single-stranded nick.
  • nucleosome does not have a fast on- and-off rate, say as it is a tightly folded part of the chromatin fiber, and it effectively blocks access for DNasel (Figure 7c).
  • hypersensitive sites HSI to HS4 are nucleosome- free in the vast majority of erythroid tissue.
  • the intervening sequences are part of an 'open' nucleosomal array, where nucleosomes compete with DNasel for access to underlying sequences.
  • the higher plateau reached in their profiles does not represent a higher proportion of entirely inaccessible sequences but all the sites in erythroid tissues receiving a single cut.
  • DNasel-hypersensitive site 4 of the human beta-globin locus control region EMBO J. 14, 106-116.

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EP02752010A 2001-05-30 2002-05-30 ACCURATE AND EFFICIENT QUANTIFICATION OF REAL-TIME PCR DNA SENSITIVITY Withdrawn EP1402072A4 (en)

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