Calibration-curve-free quantitative PCR: a quantitative method for specific nucleic acid sequences without using calibration curves

We have developed a simple quantitative method for specific nucleic acid sequences without using calibration curves. This method is based on the combined use of competitive polymerase chain reaction (PCR) and fluorescence quenching. We amplified a gene of interest (target) from DNA samples and an internal standard (competitor) with a sequence-specific fluorescent probe using PCR and measured the fluorescence intensities before and after PCR. The fluorescence of the probe is quenched on hybridization with the target by guanine bases, whereas the fluorescence is not quenched on hybridization with the competitor. Therefore, quench rate (i.e., fluorescence intensity after PCR divided by fluorescence intensity before PCR) is always proportional to the ratio of the target to the competitor. Consequently, we can calculate the ratio from quench rate without using a calibration curve and then calculate the initial copy number of the target from the ratio and the initial copy number of the competitor. We successfully quantified the copy number of a recombinant DNA of genetically modified (GM) soybean and estimated the GM soybean contents. This method will be particularly useful for rapid field tests of the specific gene contamination in samples.     

Studying copy number variations using a nanofluidic platform

Copy number variations (CNVs) in the human genome are conventionally detected using high-throughput scanning technologies, such as comparative genomic hybridization and high-density single nucleotide polymorphism (SNP) microarrays, or relatively low-throughput techniques, such as quantitative polymerase chain reaction (PCR). All these approaches are limited in resolution and can at best distinguish a twofold (or 50%) difference in copy number. We have developed a new technology to study copy numbers using a platform known as the digital array, a nanofluidic biochip capable of accurately quantitating genes of interest in DNA samples. We have evaluated the digital array's performance using a model system, to show that this technology is exquisitely sensitive, capable of differentiating as little as a 15% difference in gene copy number (or between 6 and 7 copies of a target gene). We have also analyzed commercial DNA samples for their CYP2D6 copy numbers and confirmed that our results were consistent with those obtained independently using conventional techniques. In a screening experiment with breast cancer and normal DNA samples, the ERBB2 gene was found to be amplified in about 35% of breast cancer samples. The use of the digital array enables accurate measurement of gene copy numbers and is of significant value in CNV studies.     

PCR-based quantitation of Cryptosporidium parvum in municipal water samples.

A PCR method for the quantitation of Cryptosporidium parvum oocysts in municipal drinking water samples was investigated. Quantitative PCR uses an internal standard (IS) template with unknown target numbers to compare to standards of known concentrations in a standard curve. The IS template was amplified using the same primers used to amplify a portion of a 358 bp gene fragment that encodes a repetitive oocyst wall protein in C. parvum. Municipal water samples spiked with known numbers of C. parvum oocysts were tested by quantitative PCR using the IS and the Digene SHARP Signal System Assay for PCR product detection. The absorbance readings for target DNA and IS templates versus the number of molecules of the target DNA were plotted to generate standard curves for estimating oocyst numbers. The method allowed the quantitation of oocysts from log 3 to log 5 spiked into municipal water samples.     

Determination of Transgene Copy Number in Stably Transfected Mammalian Cells by PCR-Capillary Electrophoresis Assay

The quantitative determination of transgene copy number in stably transfected mammalian cells has been traditionally estimated by Southern blot analysis. Recently, other methods have become available for appraisal of gene copy number, such as real-time PCR. Herein we describe a new method based on a fluorescently labeled PCR, followed by capillary electrophoresis. We amplified our target gene (prothrombin) and the internal control originating from genomic DNA (18S rRNA) in the same PCR tube and calculated the mean peak height ratio of the target:control gene for every cell clone sample. With this approach we identified stably transfected cell clones bearing the same transgene copy number. The results of our assay were confirmed by real-time PCR. Our method proves to be fast, low-cost, and reproducible compared with traditionally used methods. This assay can be used as a rapid screening tool for the determination of gene copy number in gene expression experiments.     

A proportional analysis method using non-kinetic real-time PCR

Prompted by increasing interest in proportional analysis of genetic types, we developed a simple assay technique for determining the ratio of a specific target gene in the total genes that can be amplified with the same PCR primer. The key feature of this method is that the following two tasks are performed in a single-tube real-time PCR system: task 1, PCR amplification of the total genes including the target using a labeled PCR primer, with concurrent monitoring of the total copy number of the PCR product; task 2, detection of the signal of the target gene at each cycle of amplification, using a labeled nucleotide probe. In principle, the ratio of the target gene to the total genes is represented by the signal detected in 'task 2' at the cycle in which the PCR product reached a prescribed copy number (assessed by 'task 1').     

Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids

We had been unsuccessful to amplify desired nucleotide sequences from various environmental DNA samples by using the inverse polymerase chain reaction (IPCR) technique, most probably because the copy numbers of target DNA sequences had been quite low. To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs). This pre-amplified IPCR (PAI-PCR) method increased the sensitivity of PCR almost 10 000 times compared with the standard IPCR in model experiments using Escherichia coli . We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts. The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.     

Quantification of the carbazole 1,9a-dioxygenase gene by real-time competitive PCR combined with co-extraction of internal standards

The fluorogenic probe assay, competitive polymerase chain reaction (PCR) and co-extraction with internal standard cells were combined to develop a rapid, sensitive, and accurate quantification method for the copy number of a target carbazole 1,9a-dioxygenase gene (carAa) and the cell number of Pseudomonas sp. strain CA10. The internal standard DNA was modified by replacement of a 20-bp long region with one for binding a specific probe in fluorogenic PCR (TaqMan). The resultant DNA fragment was similar to the corresponding region of the intact carAa gene in terms of G+C content. When used as a competitor in the PCR reaction, the internal standard DNA was distinguishable from the target carAa gene by two specific fluorogenic probes with different fluorescence labels, and was automatically detected in a single tube using the ABI7700 sequence detection system. To minimize variations in the efficiency of cell lysis and DNA extraction between the samples, the co-extraction method was combined. A mini-transposon was used to introduce competitor DNA into the genome of other pseudomonads, and the resultant construct was used as the standard cell. After the addition of a fixed amount of the internal standard cells to soil samples, total DNA was extracted (co-extraction). Using this method, the copy number of the carAa gene and the cell number of strain CA10 in soil samples could be quantified rapidly.     

PCR在HBV核酸定量检测中的应用

目的:探索一种简便定量分析系统,通过检测HBV携带者血清中的HBV X区DNA,fRNA和trRNA拷贝数,为提高隐匿性感染期低复制状态HBV检测效果提供可能。方法:从血清中提取HBV DNA和RNA,对Core区、X区DNA进行PCR扩增,使用半巢式PCR法对fRNA、trRNA在同一离心管中进行反转录并扩增,选取大小相近的质粒作为竞争模板对其进行定量。并对拉米夫定干预治疗14周前后的患者血清中HBV XDNA、Core DNA、fRNA和trRNA拷贝数进行检测与比较。所有检测结果均通过southern杂交进行验证。结果:建立了针对DNA的定量分析系统及针对RNA的RT-PCR定量分析系统,并且阐明了阿米夫定治疗前后HBV DNA和X区RNA结构和数量的变化。治疗前治疗后DNA和RNA的拷贝数均下降。Core DNA下降显著,为103-104倍,而XDNA拷贝数下降102倍。而fRNA和trRNA仅有小幅下降,为10倍左右。结论:可以通过竞争性PCR方法对血清中HBV DNA和RNA进行定量检测,以期为HBV病毒的诊断提供更充足依据。     

Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR

In transgenic plants, transgene copy number can greatly influence the expression level and genetic stability of the target gene, making estimation of transgene copy number an important area of genetically modified (GM) crop research. Transgene copy numbers are currently estimated by Southern analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials and may involve hazardous radioisotopes. We report here the development of a sensitive, high-throughput real-time (RT)-PCR technique for estimating transgene copy number in GM rice. This system uses TaqMan quantitative RT-PCR and comparison to a novel rice endogenous reference gene coding for sucrose phosphate synthase (SPS) to determine the copy numbers of the exogenous beta-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes in transgenic rice. The copy numbers of the GUS and HPT in primary rice transformants (T0) were calculated by comparing quantitative PCR results of the GUS and HPT genes with those of the internal standard, SPS. With optimized PCR conditions, we achieved significantly accurate estimates of one, two, three and four transgene copies in the T0 transformants. Furthermore, our copy number estimations of both the GUS reporter gene and the HPT selective marker gene showed that rearrangements of the T-DNA occurred more frequently than is generally believed in transgenic rice.     

Use of chromosome microdissection,the polymerase chain reaction,and dot blot hybridization to analyze double minute chromosomes

The potential usefulness of chromosome microdissertion, the polymerase chain reaction (PCR), and dot blot hybridization as a quick screening method for determining the genetic composition of double minute chromosomes (DMs) was evaluated. DMs or abrnomally banding regions (ABRs) were microdissected from from multidrug-resistant hamster cell lines and amplified with PCR using primers specific for the hamster multidrug-resistance (MDR) gene, pgp 1. The microdissected-PCR-amplified products were shown to (a) hybridize to a ³²P-labeled pCHP1 probe for the hamster MDR gene by using dot blot or Southern blot analysis and also (b) hybridize back to the chromosome region from which they were originally dissected by using fluorescent in situ hybridization. Microdissected/PCR-amplified DMs were also shown to hybridize to ABRs. When microdissected DMs and ABRs were amplified using hamster specific Alu primers, the resulting material was shown to hybridize with probes for hamster MDR and Alu. These results suggest that the DMs contained in these MDR hamster cell lines contain Alu-like sequences and the chromosome microdissection-PCR-hybridization approach might be used as a quick screening method for identifying genes amplified in DMs and ABRs in cell lines and human tumor samples.     

Timing of differential amplification of macronucleus-destined sequences during macronuclear development in the hypotrichous ciliateEuplotes crassus

The change in copy numbers of macronucleus-destined gene sequences was followed in anlagen DNA during postconjugational development inEuplotes crassus. As noted earlier, copy numbers increase during the polytene stage. During this replication process major differential amplification of different genes is not observed. Instead it is only achieved during or shortly after the fragmentation of the polytene chromosomes. This process is not totally synchronous with respect to different genes. Highly amplified genes are excised earlier than genes with a low final macronuclear copy number. Unexpectedly, the pattern of processing of the newly added oversized telomeres also appears to correlate with the degree of gene amplification. These observations are discussed in terms of a limited replication period after polytene chromosome fragmentation leading to preferential amplification of early excised genes.     

Identification of genes with correlated patterns of variations in DNA copy number and gene expression level in gastric cancer

To identify DNA copy number changes that had a direct influence on mRNA expression in gastric cancer, cDNA microarray-based comparative genomic hybridization (aCGH) and gene expression profiling were performed using 17 K cDNA microarrays. A set of 158 genes showing Pearson correlation coefficients over 0.6 between DNA copy number changes and mRNA expression level variations was selected. In an independent gene expression profiling of 60 tissue samples, the 158 genes were able to distinguish most of the normal and tumor tissues in an unsupervised hierarchical clustering, suggesting that the differential expression patterns displayed by this specific group of genes are most likely based on the gene copy number changes. Furthermore, 43 statistically significant (P<0.01) genes were selected that correctly distinguished all of the tissue samples. The copy number changes detected by aCGH can be verified by fluorescence in situ hybridization and real-time polymerase chain reaction. The selected genes include those that were previously identified as being tumor suppressors or deleted in various tumors, including GATA binding protein 4 (GATA4), monoamine oxidase A (MAOA), cyclin C (CCNC), and oncogenes including malignant fibrous histiocytoma amplified sequence 1 (MFHAS1/MASL1), high mobility group AT-hook 2 (HMGA2), PPAR binding protein (PPARBP), growth factor receptor-bound protein 7 (GRB7), and TBC1 (tre-2, BUB2, cdc16) domain family, member 1 (TBC1D1).     

Detection of novel amplicons in prostate cancer by comprehensive genomic profiling of prostate cancer cell lines using oligonucleotide-based arrayCGH

BACKGROUND: The purpose of this study was to prove the feasibility of a longmer oligonucleotide microarray platform to profile gene copy number alterations in prostate cancer cell lines and to quickly indicate novel candidate genes, which may play a role in carcinogenesis. METHODS/RESULTS AND FINDINGS: Genome-wide screening for regions of genetic gains and losses on nine prostate cancer cell lines (PC3, DU145, LNCaP, CWR22, and derived sublines) was carried out using comparative genomic hybridization on a 35,000 feature oligonucleotide microarray (arrayCGH). Compared to conventional chromosomal CGH, more deletions and small regions of gains, particularly in pericentromeric regions and regions next to the telomeres, were detected. As validation of the high-resolution of arrayCGH we further analyzed a small amplicon of 1.7 MB at 9p13.3, which was found in CWR22 and CWR22-Rv1. Increased copy number was confirmed by fluorescence in situ hybridization using the BAC clone RP11-165H19 from the amplified region comprising the two genes interleukin 11 receptor alpha (IL11-RA) and dynactin 3 (DCTN3). Using quantitative real time PCR (qPCR) we could demonstrate that IL11-RA is the gene with the highest copy number gain in the cell lines compared to DCTN3 suggesting IL11-RA to be the amplification target. Screening of 20 primary prostate carcinomas by qPCR revealed an IL11-RA copy number gain in 75% of the tumors analyzed. Gain of DCTN3 was only found in two cases together with a gain of IL11-RA. CONCLUSIONS/SIGNIFICANCE: ArrayCGH using longmer oligonucleotide microarrays is feasible for high-resolution analysis of chomosomal imbalances. Characterization of a small gained region at 9p13.3 in prostate cancer cell lines and primary prostate cancer samples by fluorescence in situ hybridization and quantitative PCR has revealed interleukin 11 receptor alpha gene as a candidate target of amplification with an amplification frequency of 75% in prostate carcinomas. Frequent amplification of IL11-RA in prostate cancer is a potential mechanism of IL11-RA overexpression in this tumor type.     

Tumor Content Chart-Assisted HER2/CEP17 Digital PCR Analysis of Gastric Cancer Biopsy Specimens

Evaluating HER2 gene amplification is an essential component of therapeutic decision-making for advanced or metastatic gastric cancer. A simple method that is applicable to small, formalin-fixed, paraffin-embedded biopsy specimens is desirable as an adjunct to or as a substitute for currently used HER2 immunohistochemistry and in situ hybridization protocols. In this study, we developed a microfluidics-based digital PCR method for determining HER2 and chromosome 17 centromere (CEP17) copy numbers and estimating tumor content ratio (TCR). The HER2/CEP17 ratio is determined by three variables—TCR and absolute copy numbers of HER2 and CEP17—by examining tumor cells; only the ratio of the latter two can be obtained by digital PCR using the whole specimen without purifying tumor cells. TCR was determined by semi-automatic image analysis. We developed a Tumor Content chart, which is a plane of rectangular coordinates consisting of HER2/CEP17 digital PCR data and TCR that delineates amplified, non-amplified, and equivocal areas. By applying this method, 44 clinical gastric cancer biopsy samples were classified as amplified (n = 13), non-amplified (n = 25), or equivocal (n = 6). By comparison, 11 samples were positive, 11 were negative, and 22 were equivocally immunohistochemistry. Thus, our novel method reduced the number of equivocal samples from 22 to 6, thereby obviating the need for confirmation by fluorescence or dual-probe in situ hybridization to < 30% of cases. Tumor content chart-assisted digital PCR analysis is also applicable to multiple sites in surgically resected tissues. These results indicate that this analysis is a useful alternative to HER2 immunohistochemistry in gastric cancers that can serve as a basis for the automated evaluation of HER2 status.     

Molecular characterisation of the t(1;15)(p22;q22) translocation in the prostate cancer cell line LNCaP

Although chromosome translocations are well-documented recurrent events in hematological malignancies and soft tissue sarcomas, their significance in carcinomas is less clear. We report here the molecular characterization of the reciprocal translocation t(1;15)(p22;q22) in the prostate carcinoma cell line, LNCaP. The chromosome 1 breakpoint was localized to a single BAC clone, RP11-290M5, by sequential FISH analysis of clones selected from the NCBI chromosome 1 map. This was further refined to a 580-bp region by Southern blot analysis. A 2.85-kb fragment spanning the der(1) breakpoint was amplified by long-range inverse PCR. The breakpoint on chromosome 1 was shown to lie between the CYR61 and the DDAH1 genes with the der(1) junctional sequence linking the CYR61 gene to the TSPAN3 (TM4SF8) gene on chromosome 15. Confirmatory PCR and FISH mapping of the der(15) showed loss of chromosome material proximal to the breakpoint on chromosome 15, containing the PSTPIP1 and RCN2 genes. On the available evidence we conclude that this translocation does not result in an in-frame gene fusion. Comparative expressed sequence hybridization (CESH) and comparative genomic hybridization (CGH) analysis, showed relative down-regulation of gene expression surrounding the breakpoint, but no gross change in genomic copy number. Real-time quantitative RT-PCR for genes around the breakpoint supported the CESH data. Therefore, here we may have revealed a gene down-regulation mechanism associated with a chromosome translocation, either through small deletion at the breakpoint or through another means of chromosome domain related gene regulation.     

RAPD isolation of a Y chromosome specific ORF in a dioecious plant, Silene latifolia.

Silene latifolia is a dioecious plant and has heteromorphic sex chromosomes: the X and Y chromosomes. The Y chromosome is the largest, and its genetic control seems to be most strict among dioecious plants. To identify the putative sex-determination elements on the Y chromosome, random amplified polymorphic DNA (RAPD) analysis was used to screen for Y chromosome specific DNA fragments, and 31 clones were successfully produced. Genomic Southern hybridization and FISH (fluorescence in situ hybridization) analyses revealed that one of the clones, #2-2, is a Y chromosome specific fragment that has a single copy on the Y chromosome. Sequence tagged site (STS)-PCR analysis also succeeded in amplifying one fragment in males and no fragments in females. Cloning and sequencing of the #2-2 flanking region using inverse PCR revealed an open reading frame (ORF) corresponding to 285 amino acids in length (ORF285), but no expression of the ORF285 gene was identified. ORF285 may be a clue to the origin of dioecy.     

In situ detection of antibiotic-resistance elements in single Bacillus cereus spores

Rapid detection of Bacillus spores is a challenging task in food and defense industries. In situ labeling of spores would be advantageous for detection by automated systems based on single-cell analysis. Determination of antibiotic-resistance genes in bacterial spores using in situ labeling has never been developed. Most of the in situ detection schemes employ techniques such as fluorescence in situ hybridization (FISH) that target the naturally amplified ribosomal RNA (rRNA). However, the majority of antibiotic-resistance genes has a plasmidic or chromosomal origin and is present in low copy numbers in the cell. The main challenge in the development of low-target in situ detection in spores is the permeabilization procedure and the signal amplification required for detection. This study presents permeabilization and in situ signal amplification protocols, using Bacillus cereus spores as a model, in order to detect antibiotic-resistance genes. The permeabilization protocol was designed based on the different layers of the Bacillus spore. Catalyzed reporter deposition (CARD)–FISH and in situ polymerase chain reaction (PCR) were used as signal amplification techniques. B. cereus was transformed with the high copy number pC194 and low copy number pMTL500Eres plasmids in order to induce resistance to chloramphenicol and erythromycin, respectively. In addition, a rifampicin-resistant B. cereus strain, conferred by a single-nucleotide polymorphism (SNP) in the chromosome, was used. Using CARD–FISH, only the high copy number plasmid pC194 was detected. On the other hand, in situ PCR gave positive results in all tested instances. This study demonstrated that it was feasible to detect antibiotic-resistance genes in Bacillus spores using in situ techniques. In addition, in situ PCR has been shown to be more sensitive and more applicable than CARD–FISH in detecting low copy targets.     

Molecular quantification of environmental DNA using microfluidics and digital PCR

Real-time PCR has been widely used to evaluate gene abundance in natural microbial habitats. However, PCR-inhibitory substances often reduce the efficiency of PCR, leading to the underestimation of target gene copy numbers. Digital PCR using microfluidics is a new approach that allows absolute quantification of DNA molecules. In this study, digital PCR was applied to environmental samples, and the effect of PCR inhibitors on DNA quantification was tested. In the control experiment using λ DNA and humic acids, underestimation of λ DNA at 1/4400 of the theoretical value was observed with 6.58ngμL(-1) humic acids. In contrast, digital PCR provided accurate quantification data with a concentration of humic acids up to 9.34ngμL(-1). The inhibitory effect of paddy field soil extract on quantification of the archaeal 16S rRNA gene was also tested. By diluting the DNA extract, quantified copy numbers from real-time PCR and digital PCR became similar, indicating that dilution was a useful way to remedy PCR inhibition. The dilution strategy was, however, not applicable to all natural environmental samples. For example, when marine subsurface sediment samples were tested the copy number of archaeal 16S rRNA genes was 1.04×10(3)copies/g-sediment by digital PCR, whereas real-time PCR only resulted in 4.64×10(2)copies/g-sediment, which was most likely due to an inhibitory effect. The data from this study demonstrated that inhibitory substances had little effect on DNA quantification using microfluidics and digital PCR, and showed the great advantages of digital PCR in accurate quantifications of DNA extracted from various microbial habitats.     

Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for Rapid Diagnosis of Sex Chromosome Aneuploidies

Sex chromosome aneuploidies occur commonly in the general population, with an incidence of 1 in 400 newborns. However, no tests specifically targeting sex chromosomes have been carried out in prenatal diagnosis or newborn screening, resulting in late recognition of these diseases. In this study, a rapid diagnostic method for sex chromosome aneuploidies was established using Quantitative Fluorescent-PCR (QF-PCR). Ten markers were included in one multiplex QF-PCR assay, including two sex determination genes (AMXY and SRY), five X-linked short tandem repeats (STRs; DXS1053, DXS981, DXS6809, DXS1187, and DXS8377), one X/Y-common STR (X22), and two autosomal STRs (D13S305 and D21S11). Retrospective tests of 70 cases with known cytogenetic results indicated that the 10-plex QF-PCR assay could well determine sex chromosome copy numbers by both allelic peak numbers and a sex chromosome dosage calculation with the autosomal STRs as internal controls. Prospective comparison with cytogenetic karyotyping on 534 cases confirmed that the 10-plex QF-PCR assay could be well employed for sex chromosome aneuploidy diagnosis in at least the Chinese Han population. This is the first QF-PCR test for the diagnosis of sex chromosome aneuploidies in the Chinese population. This test is superior to previous designs by including up to 8 sex-linked markers covering different parts of sex chromosomes as well as employing internal controls for copy number dosage calculation in a single PCR reaction. Due to simple technique and data analysis, as well as easy implementation within routine clinical services, this method is of great clinical application value and could be widely applied.     

红系特异的GFP基因在转基因小鼠中的整合和表达

应用荧光定量PCR技术对由位点控制区LCR的HS2元件和 β 珠蛋白基因启动子指导的红系特异表达绿色荧光蛋白 (GFP)基因的转基因小鼠中外源基因拷贝数进行测定 ,使用荧光显微镜和流式细胞仪检测小鼠外周血中GFP的表达水平 ,并运用荧光原位杂交技术 (FISH)确定了其中两只转基因小鼠中外源基因的整合位点 ,结果表明 :在转基因小鼠中外源基因的拷贝数各不相同且相差较大 ,而且拷贝数与GFP基因的表达量之间未呈现出相关性 ;FISH分析确定出两只转基因小鼠的外源基因整合于不同的染色体上 ;杂交信号的强弱与拷贝数的多少相一致