WO2006058395A2 - Tools and methods for the quantification of dna repeats - Google Patents
Tools and methods for the quantification of dna repeats Download PDFInfo
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- WO2006058395A2 WO2006058395A2 PCT/BE2005/000177 BE2005000177W WO2006058395A2 WO 2006058395 A2 WO2006058395 A2 WO 2006058395A2 BE 2005000177 W BE2005000177 W BE 2005000177W WO 2006058395 A2 WO2006058395 A2 WO 2006058395A2
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Definitions
- the invention relates to an assay method allowing the quantification of the copy number of a repeated nucleic acid sequence in a genetic sample.
- a genetic sample comprises the genome of a microbial, plant, animal or human subject.
- the invention provides a particular example wherein the assay is used to determine the susceptibility to disease of a subject. Such information can be used in selecting the optimal treatment for a particular diseased subject.
- DNA sequence copy number is mostly used in the development, production and evaluation of the genetic stability of cell lines. Traditionally, plasmid copy numbers of cell lines are determined by quantitating band densities produced through Southern blot analysis.
- Quantitation of band densities from Southern blots is also used in rare X-linked diseases caused by a deletion of a gene, or gene fragment, such as in Duchemme muscular dystrophy, for the determination of the carrier status in symptomless female carriers. More recently, quantitative PCR based alternatives have become available to perform copy number analysis (for example: http://www.lark.com/services/quantitative per. html). Another technique is MAPH (multiplex amplifiable probe hybridization) which was used for the determination of DNA repeats [2], however this technique is rather difficult and too laborious.
- RNA transcript
- Quantitative analysis is however most commonly used and known for the determination of transcript (RNA) levels. In most quantitative transcript assays, up to more than 10-fold differences are determined, and therefore error rates do less interfere with the result. Quantitative DNA assays may aim at the determination of less than 1-fold differences, and error rates will therefore interfere with the result, such that more precautions will be required in quantitative DNA determination than in the current quantitative RNA determination in order to obtain correct results.
- standard curves are generated using control samples. The copy number of a transcript/DNA fragment is relatively determined against another reference transcript/DNA fragment, this for different control samples. In general, a control sample is prepared in which a fixed amount of a reference fragment is mixed with a known amount of the fragment under investigation.
- control samples typically, different control samples are used, each of them having a different ratio of the concentration of the fragment under investigation to the concentration of the reference fragment, which is usually the same in all control samples.
- concentration/copy number can be deduced of the fragment under investigation in the studied samples. It is obvious that this method is prawn to pipetting errors, which can not be tolerated when small quantitative differences need to be determined. So, although the latter methods allow a fast and quantitative estimation of the DNA copy number they are prawn to such errors that they are not suitable to accurately determine the DNA copy numbers of a given sequence in a large number of samples.
- hBD1 , -2 and -3 possess chemotactic activity for immature dendritic cells and memory T-cells while hBD3 and -4 elicit monocyt chemotaxis [4].
- the hBD2, -3, -4, -5 and -6 genes are part of a 250 kb repeat region on chromosome 8p23 which is repeated 3-12 times per diploid genome [2], and therefore 3-12 gene products are found for hBD2, -3, -4, -5 and -6 in a diploid cell.
- hBD2 it has been shown that the amount of hBD2 transcripts correlate with the number of repeats.
- Cystic fibrosis is the most common lethal autosomal recessive disorder in the Caucasian population. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator channel (CFTR) gene, which is located on chromosome 7q31.3. This gene encodes a protein that is expressed in the apical membrane of exocrine epithelium cells. CFTR functions as a cAMP-induced chloride channel. CF is characterised by progressive bronchietatic lung disease, pancreatic exocrine insufficiency, chronic sinusitis and male infertility. Lung disease is the main cause of death.
- CFTR cystic fibrosis transmembrane conductance regulator channel
- F508del is the most common mutation.
- a broad spectrum of disease severity is observed between patients, even among patients with the same CFTR genotype, such as F508del/F508del, or even CF sibs.
- the CF phenotype is therefore modulated by other genetic factors, as well as environmental factors.
- Mannose-binding lectin, tumor necrosis factor alpha and transforming growth factor betai have been reported as possible modifier genes [5]. Since human /3-defensins form part of our first line of defense of the lung, they are candidate modifier genes of cystic fibrosis lung disease.
- COPD chronic bronchitis and emphysema are the most common underlying disorders.
- Emphysema is a condition of the lung characterized by abnormal, permanent enlargement of airspace distal to the terminal bronchioli accompanied by destruction of their walls and without obvious fibrosis.
- Smoking is a risk factor, but since not all smokers develop COPD, other factors are involved. Defensins are possible genetic factors involved in these diseases.
- Variable copy-number sequence means a nucleic acid sequence, which is repeated within the genetic material of a living organism. Said nucleic acid sequence can be a non-coding sequence or it can comprise one or more genes. Typically, the copy number of this repeated nucleic acid sequence varies among individuals of the same species. It is known that some sequence variation may exist between the copies of a repeated sequence, nevertheless the homology between two copies of a variable copy-number sequence should be more than 70%, more preferably more than 80% and most preferably more than 90%, for instance 95% or more.
- the present invention provides an assay kit comprising particular control constructs and methods for using said control constructs in the estimation of the copy number of variable copy-number sequences. Said methods are not affected by pipetting and dilution errors, allowing therefore the accurate determination of the copy number of the fragment under investigation. Using said methods it could be demonstrated that the copy number of the genes encoding human beta-defensins 2 to 6 could be used to predict an individual's predisposition to develop given diseases.
- Figure 1 Ligation of the cDNA of hBD2 in the pUC-18-hBD1 vector
- Figure 3 Overall distribution of FEV1 % value according to the amount of defensin genes (10 repeats or more are grouped together)
- Figure 4 Amount of defensin repeats versus least square means of the according FEV1% value with standard error.
- Figure 5 Amount of defensin repeats versus the mean FEV1 % values, linear regression analyses of the means with standard error.
- Figure 6 Relation between the copy number of the gene encoding hBD2 and the basal expression levels of hBD2
- Figure 7 Induction of beta-defensin 2 expression by TNF-alpha
- Figure 8 Variation of the copy number of the genes encoding hBD2-6 in populations having a different age range
- the invention relates to an assay method allowing the quantification of the copy number of a variable copy-number sequence within a genetic sample.
- the method of the invention has the advantage that it minimises the measurement errors due to concentration determination errors and/or pipetting errors by providing a set of at least two control constructs.
- Each control construct being an isolated nucleic acid sequence comprising the same number of copies, for instance a single copy, of a reference sequence and a different but known number of copies of a repeated nucleotide sequence.
- the reference sequence and the repeated sequence are arranged concatemerically within these control constructs.
- Said set of control constructs is used to obtain a standard curve for the calculation of the copy number of a variable copy-number sequence in a genetic sample using a quantitative assay.
- the reference sequence is located close to the variable copy-number sequence within the genome comprised in the genetic sample. Therefore, in a first object the present invention provides a set of control constructs each comprising a fixed copy number of a reference sequence and a known but variable number of copies of a repeated nucleotide sequence. Preferably the copy number of the repeated sequence in said control constructs varies between 1 and 20, more preferably between 1 and 15, for instance between 1 and 10. The invention further provides methods for using said control constructs for the quantification of the copy number of a variable copy-number sequence within a genetic sample. In a first embodiment the reference sequence comprised in each control construct corresponds to the reference sequence used to normalise the values reflecting the concentration of the variable copy-number sequence in the genetic samples.
- Each control constructs further comprises a different but known number of copies of a repeated nucleotide sequence, which corresponds to at least a part of the variable copy-number sequence of which the copy number needs to be determined in a genetic sample.
- the repeated sequence comprised in the control constructs corresponds to the part of the variable copy-number sequence in the genetic samples that is amplified by Polymerase Chain Reaction (PCR) in the quantitative assays described below.
- PCR Polymerase Chain Reaction
- These control constructs can be used in different types of quantitative PCR assays. For instance when using said control constructs in a real-time TaqManTM PCR the copy number of a variable copy-number sequence in a genetic sample can be determined as follows:
- step (v) using this relationship to calculate the copy number of the variable copy-number sequence in a genetic sample comprising an unknown copy number after determination of the Ct-ref - Ct-variable for that sample, preferably using the same primers as used in step (i).
- An alternative method for the estimation of the copy number of a variable copy-number sequence in genetic samples comprises PCR amplification of the reference and the repeated sequence followed by the quantification of the obtained PCR products.
- sequence specific primers are used whereby one of the primers for the reference sequence and one of the primers for the repeated sequence comprises a tag, for instance a biotinyl molecule, allowing the purification of the PCR products from unreacted primers.
- the other primer for the reference sequence and the other primer for the repeated sequence comprises a label, for instance a fluorescent label, allowing the detection of the PCR-product in function of its concentration.
- said primer for the reference product comprises a different label, for instance a different fluorochrome, than said primer for the repeated sequence.
- a PCR is performed with each control construct using said primers, preferably in a multiplex configuration, and after isolation of the obtained PCR-products the signal associated with the PCR-product of the repeated sequence is detected and normalised against the signal obtained for the PCR-product of the reference signal.
- Said normalised values allow calculating a standard curve indicating the relation between the normalised values and the copy number of the repeated nucleotide sequence. Using this standard curve the number of repeats of a variable copy-number sequence in a genetic sample can be extrapolated after PCR amplification using said primers and detection of the obtained PCR product of respectively the reference sequence and of the part of the variable copy-number sequence.
- a variant of this method which does not require labelling of the primers, comprises the PCR amplification, preferably in a multiplex configuration, of the reference and repeated sequence of the different control constructs. Thereafter, the PCR product of each sample is bound to a membrane (dot-blotting) or beads and the bound material is hybridised with sequences specific to the reference sequence and the repeated sequence, respectively. Alternatively, said sequences specific to the reference sequence and the repeated sequence, respectively, are, bound to a membrane (reversed-dot-blot) or beads and the PCR products are hybridised to the bound sequences. The strength of the hybridisation signal of the repeated sequence is normalised against the hybridisation signal of the reference sequence for each of the control constructs.
- each control construct does not correspond to the reference sequence used to normalise the values reflecting the concentration of the variable copy-number sequence in the genetic samples.
- Each control construct further comprises a different but known number of copies of a repeated nucleotide sequence, which differs from the variable copy-number sequence of which the copy number is to be determined in the genetic sample.
- a reference sequence and a part of the variable copy-number sequence are amplified using sequence specific primers.
- At least one primer of the reference sequence comprises a 5' adapter sequence, said sequence corresponding to at least a part of the reference sequence comprised in the control constructs.
- at least one primer of the variable copy-number sequence comprises a 5' adapter sequence, said sequence corresponding to at least a part of the repeated sequence comprised in the control constructs.
- This normalised hybridisation signal allows the extrapolation of the copy number of the variable copy number in the genetic sample after setting a standard curve using solutions of the respective control constructs.
- a sample of each control construct is hybridised with the sequences complementary to the respective adapters. It is clear that the hybridisation signal obtained with the sequence complementary to the adapter of the primer of the reference sequence reflects the concentration of the reference sequence in the control construct sample. On the other hand, the hybridisation signal obtained with the sequence complementary to the adapter of the primer of the reference sequence reflects the concentration of the repeated sequence in the control construct.
- a standard curve can be calculated indicating the relation between the normalised hybridisation values and the copy number of the repeated sequence.
- control constructs of the second embodiment allow the quantification of the copy number of the different variable copy-number sequences, using the same control constructs. Only the primers need to be customised in function of the variable copy-number sequence of interest and the selected reference sequence. However, the availability of different sets of control constructs, each set comprising a different reference sequence and repeated sequence, makes it possible to simultaneously quantify the copy number of different variable copy-number sequences in a single assay, for instance a multiplex quantitative PCR assay.
- a third embodiment of the present invention provides control constructs according to the first or second embodiment comprising multiple reference sequences and multiple repeated sequences. Such control constructs can be used to simultaneously quantify the copy number of different variable copy-number sequences in a single assay, for instance a multiplex quantitative PCR assay.
- variable copy-number sequences (more than 255 loci) in the human genome which are known to comprise sequences of which the copy number varies among individuals.
- these variable copy- number sequences are larger than 2kb and more preferably larger than 5 kb.
- the availability of these assays certainly will allow the development and implementation of genetic tests evaluating the relationship between the severity of and/or predisposition to a disease of a subject and the copy number of a given sequence in its genome.
- the invention provides a method for predicting the severity of lung disease of a patient and/or predisposition of a subject to develop lung disease based on the determination of the copy number of the variable copy- number sequence comprising the genes encoding beta-defensins 2 to 6.
- this method is used to predict the lung disease severity in CF patients. It is clear that the prediction of the potential severity of a disease in a particular patient assists the clinician in deciding on providing a preventive and/or more intensive treatment to such patient.
- the present invention relates to the use of a beta- defensin or a derivative thereof and more particularly any of the beta-defensins 2 to 6 or derivatives thereof for the preparation of a medicinaln.
- this medicinaln is used to treat CF patients having a low copy number of the variable copy-number sequence comprising the genes encoding beta-defensins 2 to 6.
- Example 1 Development of an assay system for the quantification of the copy number of the genes encoding hBD2-6 and its use in the study of the genetic basis of cystic fibrosis.
- the FEV1 values which are a measure of lung function, were taken from the clinical records of the patients. Only the disease status at a small age range was studied, i.e. the age of the patients varied between 11 and 15 years, in order to normalise for age. The FEV1 values were normalised to FEV1% according to Knudson et al. [6].
- the copy number of a gene that is part of repeat should be determined relatively to a gene that is not part of that repeat. Since that hBD1 is not part of the repeat region and located next to the repeat region containing the hBD2-6 genes, hBD1 could be used for normalisation of a gene that is part of the repeat such as hBD2.
- the reference DNA fragment should preferably be a DNA fragment next to the repeat DNA fragment, which is more likely to have a similar local DNA structure and accessibility.
- a control construct is needed containing the respective number of the repeated hBD2 fragments and 1 hBD1 fragment. Therefore, for each repeat number, we decided to construct a concatemeric vector, i.e.
- PCR was then performed with the hBD1 primers: 5 1 -TCCAAAGGAGCCAGCCTCTC-3 1 (SEQ ID No 1 ) and 5'AAAAAGTTCATTTCACTTCTGCGT C-3' (SEQ ID No 2) and hDD2 primers: 5'- CCAGCCATCAGCCATGAGGGT-3 (SEQ ID No 3) and 5 1 -TGGTTTACATGTCGCACGTC-3 1 (SEQ ID No 4).
- the PCR-product was ligated into the Sma/BAP site of pUC-18 and sequencing was performed to test if the cloning was successfull.
- hBD2 vector In order to ligate an hBD2 fragment into the pUC18 Sa/I restriction site of pUC18-hBD1 , a newhBD2 vector was made. PCR was performed with adaptor hBD2-primers containing restriction sites at their 5' end: hBD2Xall: TAGGTCGACCAGCCATCAGCCATGAGGGT-3' (SEQ ID No 5) and hBD2Xho ⁇ : ⁇ '-ACTCGAGTGGTTACATGTCGCACGTC-S' (SEQ ID NO 6). The hBD2 fragment was then cloned in PCR2.1 (Invitrogen), using the Sa/I and Xho ⁇ restriction sites.
- PCR2.1 Invitrogen
- the PCR2.1-hBD2 vector was then digested with Sa/I and Xho ⁇ .
- the insert was separated from the vector by gel electrophoreses on a 2% agarose gel and purified using the Qiagen gel extraction kit.
- the hBD2 insert was ligated in the defosforylated pUC-18-hBD1 vector and cloned in XLI blue electrocompetent cells. The insertion was controlled by PCR and sequencing.
- the plasmid DNA was obtained by extraction with nucleobond AX500.
- the subsequent constructs where made by digesting the pUC-18hBD1-hBD2 vector with Sail and ligating the hBD2-insert in the defosforylated vector ( Figure 1).
- DNA was isolated from whole blood samples using standard molecular biology procedures.
- the diploid copy number of hBD2 of each sample was determined by real time PCR with the qPCR Core kit (Eurogentec) on an ABI 7000 machine (Applied Biosystems). For each sample, a PCR was performed with the following primers and probe for hBD1 :
- the PCR reaction was performed in a 96 well clear optical reaction plates (Applied Biosystems); for each sample, 3 independent PCR reactions were performed and each of these 3 assays was even performed in duplo.
- the reaction conditions were set at 5O 0 C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60 0 C for 1 min.
- Each 96 well plate contained 6 hBD2 control constructs.
- the delta Ct value of the control constructs (Ct hBD1 - Ct hBD2) was correlated to the amount of hBD2 in each plasmid. In this way a standard curve could be constructed for each 96-well plate; which could then be used to determine the exact hBD2 copy number of the different samples under investigation.
- the diploid hBD2 copy number was determined in 135 Belgian, Czech and South-Italian CF patients homozygous for the F508del mutation. All TaqMan experiments were independently performed 3 times. On each 96-well plate, all tests were performed in duplo and contained all control constructs, so that a standard curve was made for each experiment/plate. Over the three experiments the standard deviation of the amount of repeats was less then 1.
- the table shows that a higher amount of repeats correlates with a higher FEV1% value.
- the average repeat number found in the whole population is 6.
- the results listed in Table 2 are plotted in Figure 4, and for each repeat the standard error is indicated. From 3 to 6 repeats the amount of repeats correlates strongly positively with the FEV1 % values. A more detailed look of the correlation between the amount of repeats from 3 to 6 repeats and the FEV1 % shows that this correlation seems to be linear in this range as shown in Figure 5 with a Revalue of 0.981. Moreover, the 0.95 confidence intervals do not overlap. In the smaller repeat range, one repeat unit more or less has already a significant effect on the FEV1% value.
- EXAMPLE 2 Effect of the copy number of the genes encoding hBD2-6 in a study on the genetic basis of COPD
- the diploid hBD2 copy number was determined in 69 Belgian smoking control patients and 44 Belgian COPD patients. All TaqMan experiments were independently performed 3 times. On each 96-well plate, all tests were performed in duplo and contained all control constructs, so that a standard curve was made for each experiment/plate. Over the three experiments the standard deviation of the amount of repeats was less then 1.
- Example 3 Correlation of hBD2 transcript levels with the number of hBD2 repeats
- Nasal polyps were obtained from about 50 patients having rhinosinusitus, in whom polyps were removed for medical reasons by a surgical intervention. From these tissue samples, monolayers of epithelial cells were grown as described before [7].
- tissue samples were placed in Dulbecco's Modified Eagles Medium (DMEM) (Invitrogen), supplemented with 100 U of penicillin (PE) (Invitrogen) and 100 mg/ml streptomycin (ST) (Invitrogen), before transportation to the laboratory.
- DMEM Dulbecco's Modified Eagles Medium
- PE penicillin
- ST streptomycin
- the samples were cut into small pieces (using a sterile surgical blade) and placed on a tumble mill in a container with a solution of pronase (1 mg/ml) (Sigma) for 16-24 h in a cold chamber (4°C) to enable enzymatic dissociation of the epithelium.
- the second part of the cells was resuspended in 10 ml Versene (Invitrogen) and after centrifugation resuspended in 1.5 ml Trypsine (Invitrogen). After 7 minutes, the cells were washed in 10 ml DMEM plus PEST plus 10% FCS. After centrifugation (70Og), the pellet was resuspended in 3 ml 10% GCS in DMEM F12 plus PEST plus 5% DMSO and frozen in liquid nitrogen for later use, such as re-culturing.
- RNA (1 ⁇ g) was reverse transcribed into cDNA using superscript reverse transcriptase.
- a delta Ct value of n equals a copy number variation of 2 n .
- the variation in expression was then calculated by 2 cthBD1"cthBD2 and evaluated against the number of the repeats found at the genomic level.
- TNF ⁇ Tumor necrosis factor alpha
- PBS phosphate buffered saline
- BSA bovine serum albumin
- This TNF ⁇ stock solution was 100x diluted in DMEM-F12 plus PEST.
- the upper layer of the cells was washed with PBS before applying 100 ⁇ l TNF-alpha solution to the cells.
- the 12- well plates were incubated in a 5% CO 2 incubator for different time periods. After incubation, the epithelial cells were washed 3 times with PBS and the RNA was extracted from the cells with an RNA extraction kit (Qiagen). DNA was removed during RNA extraction with a
- RNAse-free DNAse kit Qiagen. The RNA was stored at -7O 0 C.
- RNA (1 ⁇ g) was reverse transcribed into cDNA using superscript reverse transcriptase.
- the relative amount of IL-S and hBD2 was determined with real time PCR on the ABI 7000.
- IL-8 was used as a control.
- IL-8, hBD2, and the housekeeping gene human porphobilinogen deaminase (hPBGD) were analysed.
- the obtained Ct values i.e. the number of PCR amplification rounds required to reach a certain threshold amount of amplification products in the exponential phase of the PCR reaction, is directly correlated to the amount of target mRNA. The higher the amount of target mRNA (transcribed into cDNA) in the sample, the lower the Ct value.
- ⁇ Ct represents the relative amount of transcripts of the gene under analysis in the sample.
- a change in ⁇ Ct n represents a change in expression of the gene under study with a factor 2 n .
- a negative control was included in each experiment.
- hBD2 and IL-8 were evaluated after induction with 10 ng TNF-alpha. It was previously shown that after 12 hours of induction, the expression of hBD2 and IL-8 is significantly induced in epithelial tissue [8].
- hBD2 because we observed a correlation between the number of repeats and the basal expression of this defensin.
- IL-8 in the study as a control for the experiment. TNF-alpha induces IL-8 expression, but it is expected that IL-8 expression is not altered by the number of defensin repeats.
- Example 4 Variation of the copy number of the genes encoding hBD2-6 in populations having a different age range
- the number of defensin repeats was determined in two control groups that varied in age range. A different distribution of repeats was found in the two controls groups, i.e. in the older control group, a lower proportion of individuals carried a high defensin repeat number.
- a possible explanation is that a high repeat number of this defensin region is deleterious in common complex diseases, e.g. lung diseases, auto-immune diseases, amongst others, and these individuals are lost because they die earlier and therefore are underrepresented in the older group of individuals (Figure 8). This is an indication that defensins may be involved in other diseases than in CF, and not necessarily in the same way as in a pediatric disease such as CF.
- Table 1 F508del/F508del CF population: Origin of the patients and the corresponding mean FEVl % value and mean amount of repeats.
- Table 3 Division of the data set into three groups with the corresponding FEV1% values and the standard deviation
Abstract
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DE602005007263T DE602005007263D1 (en) | 2004-11-30 | 2005-11-30 | TOOLS AND METHOD FOR QUANTITATING DNA REPEAT SEQUENCES |
US11/791,711 US20080118925A1 (en) | 2004-11-30 | 2005-11-30 | Tools and Methods for the Quantification of Dna Repeats |
EP05817922A EP1819834B8 (en) | 2004-11-30 | 2005-11-30 | Tools and methods for the quantification of dna repeats |
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WO2007129000A2 (en) * | 2006-04-12 | 2007-11-15 | Medical Research Council | Method for determining copy number |
WO2013093005A1 (en) | 2011-12-22 | 2013-06-27 | Centre National De La Recherche Scientifique (Cnrs) | Method of dna detection and quantification by single-molecule hybridization and manipulation |
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WO2011143611A2 (en) | 2010-05-14 | 2011-11-17 | Life Technologies Corporation | Karyotyping assay |
EP3789022A1 (en) | 2012-12-27 | 2021-03-10 | Massachusetts Eye & Ear Infirmary | Treatment of rhinosinusitis with p-glycoprotein inhibitors |
US11408900B2 (en) | 2016-01-15 | 2022-08-09 | Massachusetts Eye And Ear Infirmary | Secreted P-glycoprotein is a non-invasive biomarker of chronic rhinosinusitis |
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Title |
---|
KE L D ET AL: "A reliability test of standard-based quantitative PCR: exogenous vs endogenous standards" MOLECULAR AND CELLULAR PROBES, vol. 14, no. 2, April 2000 (2000-04), pages 127-135, XP004435437 ISSN: 0890-8508 * |
LALLEMAND F ET AL: "Quantitative analysis of human herpesvirus 8 viral load using a real-time PCR assay" JOURNAL OF CLINICAL MICROBIOLOGY, vol. 38, no. 4, April 2000 (2000-04), pages 1404-1408, XP002909016 ISSN: 0095-1137 * |
PARDIGOL ANDREAS ET AL: "A simple procedure for quantification of genetically modified organisms using hybrid amplicon standards." EUROPEAN FOOD RESEARCH AND TECHNOLOGY, vol. 216, no. 5, May 2003 (2003-05), pages 412-420, XP002374753 ISSN: 1438-2377 * |
TAVERNIERS ISABEL ET AL: "Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods." ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 378, no. 5, March 2004 (2004-03), pages 1198-1207, XP002374754 ISSN: 1618-2642 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2007129000A2 (en) * | 2006-04-12 | 2007-11-15 | Medical Research Council | Method for determining copy number |
WO2007129000A3 (en) * | 2006-04-12 | 2008-03-13 | Medical Res Council | Method for determining copy number |
WO2013093005A1 (en) | 2011-12-22 | 2013-06-27 | Centre National De La Recherche Scientifique (Cnrs) | Method of dna detection and quantification by single-molecule hybridization and manipulation |
US9738924B2 (en) | 2011-12-22 | 2017-08-22 | Centre National De La Recherche Scientifique (Cnrs) | Method of DNA detection and quantification by single-molecule hybridization and manipulation |
Also Published As
Publication number | Publication date |
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EP1819834A2 (en) | 2007-08-22 |
WO2006058395A3 (en) | 2006-09-14 |
DE602005007263D1 (en) | 2008-07-10 |
US20080118925A1 (en) | 2008-05-22 |
EP1819834B1 (en) | 2008-05-28 |
EP1819834B8 (en) | 2008-10-29 |
GB0426188D0 (en) | 2004-12-29 |
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