Development and evaluation of real competitive PCR for high-throughput quantitative applications

Real competitive PCR (rcPCR) has been shown to have high sensitivity, reproducibility, and high-throughput potential. We describe further development and evaluation of this methodology as a tool for measuring nucleic acid abundance within a cell. Modifications to the original protocol allow analysis of gene expression levels using standard conditions regardless of mRNA abundance and assay type, thereby increasing throughput and ease of reaction setup while decreasing optimization time. In addition, we have developed a software package, TITAN, to automatically analyze the results. The details are relevant to researchers performing competitive PCR using any detection technique. The effectiveness of the described developments is demonstrated using 12 genes known to have differential expression in cell lines grown under normal and hypoxic conditions. Quantitative and qualitative comparisons to real-time PCR are presented. It is also demonstrated that the technique is capable of detecting submicroscopic chromosomal DNA deletions.     

Detection of rare K-ras codon 12 mutations using allele-specific competitive blocker PCR

Allele-specific competitive blocker PCR (ACB-PCR) is a sensitive allele-specific amplification method in which preferential amplification of the mutant allele occurs by using a primer that has more mismatches to the wild-type allele than to the mutant allele (mutant-specific primer, MSP). Additionally, a non-extendable primer with more mismatches to the mutant allele than to the wild-type allele (blocker primer, BP) competes with the MSP for binding to the wild-type allele, thereby reducing background amplification from the wild-type allele. ACB-PCR primer design is largely dependent upon the basepair substitution being measured, making it unclear if this method is broadly applicable. In an earlier study, an H-ras codon 61 CAA-->AAA mutation had been detected by ACB-PCR at a sensitivity of 10(-5). In this study, ACB-PCR was applied to two human K-ras codon 12 mutations: GGT-->GTT and GGT-->GAT. The method was optimized by systematically altering the concentrations of Perfect Match PCR Enhancer, MSP, BP, and dNTPs. For each mutation, mutant fractions as low as 10(-5) were detected, indicating that this assay can be used on a variety of base substitution mutations. In addition, the results suggest that the 3'-terminal mismatches between the MSP and wild-type allele may be used to predict the ACB-PCR conditions that will be appropriate for the detection of other base substitution mutations. The range of concentrations for each of these components is narrow, making this method relatively easy to apply to additional mutational targets.     

Rapid and quantitative method of allele-specific DNA methylation analysis

Several biological phenomena depend on differential methylation of chromosomal strands. While understanding the role of these processes requires information on allele-specific methylation, the available methodologies are not quantitative or labor-intensive. We describe a novel, rapid method to quantitate allele-specific DNA methylation based on the combination of bisulfite PCR and Pyrosequencing. In this method, DNA is first treated with sodium bisulfite, which converts cytosine but not 5-methylcytosine to uracil. Genes of interest are subsequently amplified using PCR. Allele-specific methylation can then be determined by pyrosequencing each allele individually using sequencing primers that incorporate single nucleotide polymorphisms (SNPs) that allow differentiation between the two parental alleles. This allele-specific methylation methodology can potentially afford quantitative analyses relevant to the regulation of X chromosome inactivation, allele-specific expression of genes in the immune system, repetitive elements, and genomic imprinting. As an illustration of our new method, we quantitated allele-specific methylation of the differentially methylated region of the H19 gene, which is imprinted. Although we could reliably determine allele-specific methylation with our technique, additional studies will be required to confirm the ability of our assay to measure loss of imprinting.     

Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment

A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR.     

Detection and quantification of copper-denitrifying bacteria by quantitative competitive PCR

We developed a quantitative competitive PCR (QC-PCR) system to detect and quantify copper-denitrifying bacteria in environmental samples. The primers were specific to copper-dependent nitrite reductase gene (nirK). We were able to detect about 200 copeis of nirK in the presence of abundant non-specific target DNA and about 1.2 x 10(3)Pseudomonas sp. G-179 cells from one gram of sterilized soil by PCR amplification. A 312-bp nirK internal standard (IS) was constructed, which showed very similar amplification efficiency with the target nirKfragment (349 bp) over 4 orders of magnitude (10(3)-10(6)). The accuracy of this system was evaluated by quantifying various known amount of nirK DNA. The linear regressions were obtained with a R(2) of 0.9867 for 10(3)copies of nirK, 0.9917 for 10(4) copies of nirK, 0.9899 for 10(5) copies of nirK and 0.9846 for 10(6) copies of nirK. A high correlation between measured nirK and calculated nirK (slope of 1.0398, R(2)=0.9992) demonstrated that an accurate measurement could be achieved with this system. Using this method, we quantified nirK in several A-horizon and stream sediment samples from eastern Tennessee. In general, the abundance of nirK was in the range of 10(8)-10(9) copies g soil(-1) dry weight. The nirK content in the soil samples appeared correlated with NH(4)(N) content in the soil. The activities of copper-denitrifying bacteria were evaluated by quantifying cDNA of nirK. In most of sample examined, the content of nirK cDNA was less than 10(5) copies g soil(-1) dry weight. Higher nirK cDNA content (>10(6) copies g soil(-1) dry weight) was detected from both sediment samples at Rattlebox Creek and the Walker Branch West Ridge. Although the stream sediment samples at the Walker Branch West Ridge contained less half of the nirK gene content as compared to A-horizon sample, the activities of copper-denitrifying bacteria were almost 600 times higher than in the A-horizon sample.     

Comparison of competitive and positive control-based PCR quantitative procedures coupled with end point detection

Two PCR methods using internal standards, coupled with our sandwich nonisotopic enzyme-linked oligosorbent assay (ELOSA) in microtiter plate format, were developed for quantitation of human immunodeficiency virus type 1 (HIV-1) provirus. We present an overview of both methodologies focusing on two major features, i.e., the conditions of equivalency of replication efficiency and the definition of criteria of acceptance validating a result. Quantitative competitive PCR (QC-PCR) was based on the coamplification of the HIV-1 nef gene with different amounts of a pNEFmut plasmid that contains the nef gene with different amounts of a pNEFmut plasmid that contains the nef region but with mutations in the capture probe recognition region. The NEF wild-type (NEF) and the NEF mimic (NEFmut) amplification products were differentiated in ELOSA. NEFmut OD to NEF OD ratios were plotted against the number of mimic copies, and the deduced linear curve permitted quantitation of HIV-I copy number. Internally controlled PCR (IC-PCR) was based on coamplification of the HIV-1 nef gene with an internal endogenous standard, the ras gene, as a positive control of amplification. HIV-1 copy number was determined using external standard of known amounts of HIV-1 DNA. We address the advantages as well as the limitations of individuals protocols and discuss future improvements of quantitative amplification process.     

Identification of self-incompatibility genotypes of almond by allele-specific PCR analysis

In almond, gametophytic self-incompatibility is controlled by a single multiallelic locus (S-locus). In styles, the products of S-alleles are ribonucleases, the S-RNases. Cultivated almond in California have four predominant S-alleles (S ^a, S ^b, S ^c, S ^d). We previously reported the cDNA cloning of three of these alleles, namely S ^b, S ^c and S ^d. In this paper we report the cloning and DNA sequence analysis of the S ^a allele. The S^a-RNase displays approximately 55% similarity at the amino-acid level with other almond S-RNases (S^b, S^c, and S^d) and this similarity was lower than that observed among the S^b, S^c and S^d-RNases. Using the cDNA sequence, a PCR-based identification system using genomic DNA was developed for each of the S-RNase alleles. Five almond cultivars with known self-incompatibility (SI) geno-types were analyzed. Common sequences among four S-alleles were used to create four primers, which, when used as sets, amplify DNA bands of unique size that corresponded to each of the four almond S-alleles; S ^a (602 bp), S ^b (1083 bp), S ^c (221 bp) and S ^d (343 bp). All PCR products obtained from genomic DNA isolated from the five almond cultivars were cloned and their DNA sequence obtained. The nucleotide sequence of these genomic DNA fragments matched the corresponding S-allele cDNA sequence in every case. The amplified products obtained for the S ^a- and S ^b-alleles were both longer than that expected for the coding region, revealing the presence of an intron of 84 bp in the S ^a-allele and 556 bp in the S ^b-allele. Both introns are present within the site of the hypervariable region common in S-RNases from the Rosaceae family and which may be important for S specificity. The exon portions of the genomic DNA sequences were completely consistent with the cDNA sequence of the corresponding S-allele. A useful application of these primers would be to identify the S-genotype of progeny in a breeding program, new varieties in an almond nursery, or new grower selections at the seedling stage. Received: 21 June 1999 / Accepted: 15 November 1999     

A competitive PCR assay for quantitative detection of Neospora caninum

A quantitative-competitive PCR (QC-PCR) assay was developed for measurement of Neospora caninum levels in the tissues of infected animals. A molecule was synthesised for use in PCR as a competitor to the target Neospora-specific Nc5 genomic sequence. The assay was used to evaluate the relative level of parasites in the brain and lungs of mouse pups in a model of vertical transmission of N. caninum. Infection on day 11 of gestation resulted in similar levels of parasites in all offspring. The assay should be useful in evaluation of vaccines against Neospora infection. Incorporation of the competitor molecule in the detection assay also provides a control for PCR failure and facilitates identification of truly negative samples.     

Analysis of copy number variation using quantitative interspecies competitive PCR

Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.     

Adapter-tagged competitive PCR (ATAC-PCR)--a high-throughput quantitative PCR method for microarray validation

Adapter-tagged competitive PCR (ATAC-PCR) is an advanced version of competitive quantitative PCR that is characterized by the addition of unique adapters to cDNA derived from each sample RNA. Using multiple adapters, we can accurately measure the relative expression ratios of many samples, with a calibration curve obtained from internal standards included in the same reaction. ATAC-PCR can identify differences in gene expression as small as twofold, even from very small amounts of sample RNA. This technique is suitable for confirming results obtained with cDNA microarrays or differential display, and it can process more than a thousand of genes per day when used in conjunction with a capillary DNA sequencer.     

Assignment of SNP allelic configuration in polyploids using competitive allele-specific PCR: application to citrus triploid progeny

Background

Polyploidy is a major component of eukaryote evolution. Estimation of allele copy numbers for molecular markers has long been considered a challenge for polyploid species, while this process is essential for most genetic research. With the increasing availability and whole-genome coverage of single nucleotide polymorphism (SNP) markers, it is essential to implement a versatile SNP genotyping method to assign allelic configuration efficiently in polyploids.

Scope

This work evaluates the usefulness of the KASPar method, based on competitive allele-specific PCR, for the assignment of SNP allelic configuration. Citrus was chosen as a model because of its economic importance, the ongoing worldwide polyploidy manipulation projects for cultivar and rootstock breeding, and the increasing availability of SNP markers.

Conclusions

Fifteen SNP markers were successfully designed that produced clear allele signals that were in agreement with previous genotyping results at the diploid level. The analysis of DNA mixes between two haploid lines (Clementine and pummelo) at 13 different ratios revealed a very high correlation (average = 0·9796; s.d. = 0·0094) between the allele ratio and two parameters [θ angle = tan⁻¹ (y/x) and y′ = y/(x + y)] derived from the two normalized allele signals (x and y) provided by KASPar. Separated cluster analysis and analysis of variance (ANOVA) from mixed DNA simulating triploid and tetraploid hybrids provided 99·71 % correct allelic configuration. Moreover, triploid populations arising from 2n gametes and interploid crosses were easily genotyped and provided useful genetic information. This work demonstrates that the KASPar SNP genotyping technique is an efficient way to assign heterozygous allelic configurations within polyploid populations. This method is accurate, simple and cost-effective. Moreover, it may be useful for quantitative studies, such as relative allele-specific expression analysis and bulk segregant analysis.     

Simultaneous quantitative detection of relevant biomarkers in breast cancer by quantitative real-time PCR

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.     

Real-time quantitative PCR assay for analysis of platelet glycoprotein IIIa gene expression

A quantitative detection assay for analysis of platelet glycoprotein GPIIIa gene expression is presented. The assay uses two fluorescently labeled TaqMan MGB probes to detect the polymorphic site in GPIIIa nucleotide sequence, leading to antigens HPA-1a and HPA-1b. In order to avoid the influence of DNA contamination on RNA quantification, a forward primer was constructed to span an exon-exon junction. The assay is therefore applicable to expression studies also in samples containing only a small amount of contaminating DNA. To standardize the amount of sample cDNA added to the reaction, amplification of endogenous control 18SrRNA was included in a separate well. The amplification validation experiment showed a high real-time PCR efficiency for HPA-1a, HPA-1b and 18SrRNA. Relative quantification was therefore performed using the comparative C(T) method. The assay was optimized on a reversely transcribed total RNA from platelets, and the specificity rate was determined by sequencing. The amount of cDNA at which amplification was still clearly detectable was 5 ng. This newly developed real-time quantitative PCR assay is a sensitive, reproducible and reliable method. It is suitable for studying different stages of megakaryopoiesis, monitoring molecular alteration in defective platelets and determining differences in the GPIIIa expression level between normal and pathological megakaryocytic differentiation pathways.