Input Copy Number Research Articles

Comparable b2a2/e13a2 and b3a2/e14a2 Reporting in BCR-ABL Assay When Calibrated By Who Is and By IVT-RNA Copy Number

Introduction: Xpert® BCR-ABL Ultra, automated cartridge-based assay for monitoring BCR-ABL transcript levels, is calibrated by WHO IS to standardize the %reporting for both b2a2/e13a2 BCR-ABL(e13a2) and b3a2/e14a2 BCR-ABL(e14a2) relative to control ABL gene in peripheral blood of patients as a standard management of Chronic Myeloid Leukemia (CML) [1]. Studies have shown differences in patients carrying e13a2 or e14a2 in molecular response to Tyrosine Kinase Inhibitor (TKI) treatment [2-4]. Therefore, it is necessary to understand if there is %reporting differences between two transcripts calibrated by WHO IS[5] and explore the calibration method using breakpoint specific RNA input copy number (CN) for accurate TKI treatment monitoring. Objectives: To establish % reporting method calculated with known CN of e13a2, e14a2 and ABL IVT-RNA within a wide CN range and compare % e13a2 and % e14a2 to WHO IS quantification system. Methods: Three IVT-RNAs, containing e13a2-ABL-BCR, e14a2-ABL-BCR and ABL-BCR, were synthesized as shown in Figure 1. Serial diluted e13a2 RNA, e14a2 RNA and ABL RNA panels were tested on Xpert BCR-ABL Ultra assay to generate individual standard curve of e13a2 Ct, e14a2 Ct and ABL Ct as a function of CN for % reporting calculation. Four levels of IVT-RNA panels with same CN of e13a2 and e14a2 were tested for breakpoint specific % reporting comparison. K562(e14a2), BV173 (e13a2) cell lysates and CML clinical samples carrying e13a2 or e14a2 transcripts were tested to evaluate the %CN reporting compared to WHO IS. Result: Good linearity demonstrated in Ct vs CN input for e13a2, e14a2 and ABL IVT-RNA (Figure2) with comparable efficiency (E) between e13a2 (E=0.992) and e14a2 (E=0.986). %e13a2 reporting was ~1.50-fold (by WHO IS) and ~1.46-fold (by %CN) higher than %e14a2, by testing IVT-RNA panel (Table1). Minor differences in %reporting observed between % CN and WHO IS for e13a2 (84.5%~110.8%) vs e14a2 (82.5%~89.5%) from cell lysates (Table2) and e13a2 (92.6%~105.5%) vs e14a2 (88.1%~92.2%) from clinical samples (Table3). Conclusions: A %CN e13a2/ABL and e14a2/ABL reporting method with known CN of IVT-RNAs has been successfully established in Xpert BCR-ABL Ultra. Both WHO IS and %CN showed very minor differences between e13a2 and e14a2 for % reporting. %CN method demonstrated comparable %reporting to WHO IS in Xpert BCR-ABL Ultra. Recent Publications 1. Dominy KM, Simon IM, Khorashad JS (2021) Evaluation of Xpert® BCR-ABL Ultra for the confirmation of BCR-ABL1 international scale conversion factors for the molecular monitoring of chronic myeloid leukaemia, Int J Lab Hematol, Feb;43(1):e31-e34. 2. Hanfstein B, Lauseker M, Hehlmann R, Saussele S, Erben P, Dietz C (2014) Distinct characteristics of e13a2 versus e14a2 BCR-ABL1 driven chronic myeloid leukemia under first-line therapy with imatinib. Haematologica.99:1441-7. 3. Jain P, Kantarjian H, Patel KP, Gonzalez GN, Luthra R, Shamanna RK (2016) Impact of BCR-ABL transcript type on outcome in patients with chronic-phase CML treated with tyrosine kinase inhibitors. Blood.127:1269-75. 4. Castagnetti F, Gugliotta G, Breccia M, Iurlo A, Levato L, Albano F (2017) The BCR-ABL1 transcript type influences response and outcome in Philadelphia chromosome-positive chronic myeloid leukemia patients treated frontline with imatinib. Am J Hematol. 92:797-805. 5. Matthew S, White HE, Hana Z, Nicholas C.P C (2022) Impact of BCR::ABL1 transcript type on RT-qPCR amplification performance and molecular response to therapy. Leukemia 36(7):1-8

Reducing Bias and Quantifying Uncertainty in Fluorescence Produced by PCR

We present a new approach for relating nucleic-acid content to fluorescence in a real-time Polymerase Chain Reaction (PCR) assay. By coupling a two-type branching process for PCR with a fluorescence analog of Beer’s Law, the approach reduces bias and quantifies uncertainty in fluorescence. As the two-type branching process distinguishes between complementary strands of DNA, it allows for a stoichiometric description of reactions between fluorescent probes and DNA and can capture the initial conditions encountered in assays targeting RNA. Analysis of the expected copy-number identifies additional dynamics that occur at short times (or, equivalently, low cycle numbers), while investigation of the variance reveals the contributions from liquid volume transfer, imperfect amplification, and strand-specific amplification (i.e., if one strand is synthesized more efficiently than its complement). Linking the branching process to fluorescence by the Beer’s Law analog allows for an a priori description of background fluorescence. It also enables uncertainty quantification (UQ) in fluorescence which, in turn, leads to analytical relationships between amplification efficiency (probability) and limit of detection. This work sets the stage for UQ-PCR, where both the input copy-number and its uncertainty are quantified from fluorescence kinetics.

Nanoscale Force-Mapping-Based Quantification of Low-Abundance Methylated DNA.

Methylation changes at cytosine-guanine dinucleotide (CpG) sites in genes are closely related to cancer development. Thus, detection and quantification of low-abundance methylated DNA is critical for early diagnosis. Here, we report an atomic force microscopy (AFM)-based quantification method for DNA that contains methyl-CpG at a specific site, without any treatment to the target DNA such as chemical labeling, fluorescence tagging, or amplification. We employed AFM-tip-tethered methyl-CpG-binding proteins to probe surface-captured methylated DNA. We observed a linear correlation (R2 = 0.982) between the input copy number and detected copy number, in the low copy number regime (10 or fewer; subattomolar concentrations). For a mixture of methylated and nonmethylated DNA that resembles clinical samples, we were still able to quantify the methylated DNA. These results highlight the potential of our force-mapping-based quantification method for wide applications in early detection of diseases associated with methylated DNA.

Development of Reference Control Material for the Evaluation of the Cepheid Prototype * Xpert® BCR-ABL P190 Ultra Assay

Background: Patients with Philadelphia+ Acute Lymphoid Leukemia (Ph+ ALL) respond to the same tyrosine kinase inhibitors (TKIs) that work against Chronic Myeloid Leukemia (CML). However, the dominant BCR-ABL breakpoint is the e1a2 (p190) minor transcript with the e13a2/e14a2 (p210) major transcripts, accounting for 20-30% of the ALL patients, together with <5% of the CML patients. Currently, unlike the p210 major transcripts for CML, there are no International Standards available for the quantitation of the e1a2 p190 transcript for both CML and ALL. This poses a challenge when developing a quantitative assay to monitor the e1a2 p190 transcript levels. Objectives: Implementation of a certified plasmid DNA (pDNA) reference material through copy number (CN) calibration was used to develop a primary control pDNA at Cepheid. The reference pDNA contains ABL and BCR-ABL b3a2-p210 (IRMM_ERM-AD623 BCR-ABL Calibrator) for standardization of ABL and BCR-ABL e1a2-p190 quantifications. This process allows generation of in-house secondary RNA control panel, for calibration and standardization of the Xpert BCR-ABL p190 Ultra assay to the WHO IS (NIBSC-09/138). Method: Titration studies were performed with IRMM's pDNA to derive a CN calibration curve of the ABL control gene relative to ABL Cycle Threshold (Ct), to assign CN to Cepheid's pDNA containing e1a2 target gene and ABL control gene in a 1:1 ratio. The obtained CN information for Cepheid's pDNA was then used to generate corresponding ABL Ct and e1a2 Ct in a simulated biological matrix, followed by the assignment of CN to the in-house RNA control material. A 5-log concentration range of a malignant BCR-ABL e1a2 positive ALL cell line, SUP-B15, is used as an interim in-house control material with an assigned % BCR-ABL e1a2/ABL value to evaluate the assay's linearity/dynamic range performance. Results: Through the ABL calibration curve generated from IRMM reference material, CN of Cepheid's e1a2:ABL pDNA were assigned. The ABL Ct was generated using IRMM reference material with a 6-log dynamic range of ABL CN, slope of -1.41, R2=0.9924 (Figure 1). Using the ABL Ct obtained from the Cepheid's pDNA construct, allowed us to link Cepheid's pDNA ABL CN to IRMM's, thus linking e1a2 CN to ABL CN, demonstrated in a 5-log dynamic range of ABL and e1a2 CN. Similar slopes of -1.491, R2=0.9972 and -1.464, R2=0.9966 were obtained from Cepheid's pDNA between input CN and output Ct for e1a2 and ABL, respectively (Figure 2). Finally, a 5-log dynamic range of ∆Ct (e1a2-ABL) was generated from SUP-B15 cells, yielding slope of -1.427, R2=0.9917 (Figure 3), allowing assignment of %BCR-ABL e1a2/ABL value. Conclusion: In conclusion, implementing a certified pDNA reference material (IRMM) allowed linkage of known ABL CN to Cepheid's pDNA. Preliminary data from SUP-B15 cells suggested that we achieved the goal of generating a control material with assigned e1a2 CN. * Prototype test. Not for use in diagnostic procedures. Not reviewed by any regulatory body. Disclosures No relevant conflicts of interest to declare.

Prospect and Competence of Quantitative Methods via Real-time PCR in a Comparative Manner: An Experimental Review of Current Methods

Background: There are numerous approaches dealing with relative and absolute quantitation. The methods differ in their efficiency assumption and applicability. Objective: Current methodologies and rations used in qPCR quantification were compared in an experimental study of transgenic copy number determination of a monoclonal antibody Daclizumab. Methods: With an inter and intra-methodical view, variations in relative and absolute quantification strategies were discretely extracted and compared to one another. Results: In relative quantification, six methods were studied and the ratios were computed relative to Glucagon as internal control. For Absolute quantification, the calculations were based on standard curve. Relative quantification considers the relative changes in expression levels while Absolute quantification relates the PCR signal to input copy number with a calibration curve. Conclusion: The observed unevenness of the ratios in Relative approach pointed mainly to the efficiency changes and its calculation formula. Whereas results in Absolute approach strategies showed homogeneity which indicates the consistency of the calculation method.

Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) \u2013 A New Versatile Isothermal Amplification Method

The growing demand for cost-effective nucleic acid detection assays leads to an increasing number of different isothermal amplification reaction methods. However, all of the most efficient methods suffer from highly complex assay conditions due to the use of complicated primer sets and/or auxiliary enzymes. The present study describes the application of a new linker moiety that can be incorporated between a primer and a secondary target binding site which can act both as a block to polymerase extension as well as a hinge for refolding. This novel “hinge-primer” approach results in an efficient regeneration of the primer binding site and thus improves the strand-displacement and amplification process under isothermal conditions. Our investigations revealed that the reaction with forward and reverse hinge-primer including an abasic site is very efficient. The assay complexity can be reduced by combining the hinge-primer with a corresponding linear primer. Furthermore, the reaction speed can be increased by reducing the length of the amplified target sequence. We tested the sensitivity down to 104 copies and found a linear correlation between reaction time and input copy number. Our approach overcomes the usually cumbersome primer-design and extends the range of isothermal amplification methods using a polymerase with strand-displacement activity.

"Deep" sequencing accuracy and reproducibility using Roche/454 technology for inferring co-receptor usage in HIV-1.

Next generation, “deep”, sequencing has increasing applications both clinically and in disparate fields of research. This study investigates the accuracy and reproducibility of “deep” sequencing as applied to co-receptor prediction using the V3 loop of Human Immunodeficiency Virus-1. Despite increasing use in HIV co-receptor prediction, the accuracy and reproducibility of deep sequencing technology, and the factors which can affect it, have received only a limited level of investigation. To accomplish this, repeated deep sequencing results were generated using the Roche GS-FLX (454) from a number of sources including a non-homogeneous clinical sample (N = 47 replicates over 18 deep sequencing runs), and a large clinical cohort from the MOTIVATE and A400129 studies (N = 1521). For repeated measurements of a non-homogeneous clinical sample, increasing input copy number both decreased variance in the measured proportion of non-R5 using virus (p<<0.001 and 0.02 for single replicates and triplicates respectively) and increased measured viral diversity (p<0.001; multiple measures). Detection of sequences with a mean abundance less than 1% abundance showed a 2 fold increase in median coefficient of variation (CV) in repeated measurements of a non-homogeneous clinical sample, and a 2.7 fold increase in CV in the MOTIVATE/A400129 dataset compared to sequences with ≥1% abundance. An unexpected source of error included read position, with low accuracy reads occurring more frequently towards the edge of sequencing regions (p<<0.001). Overall, the primary source of variability was sampling error caused by low input copy number/minority species prevalence, though other sources of error including sequence intrinsic, temporal, and read-position related errors were detected.

Factors Influencing the Sensitivity and Specificity of Conventional Sequencing in Human Immunodeficiency Virus Type 1 Tropism Testing

Human immunodeficiency virus type 1 (HIV-1) V3 loop sequence can be used to infer viral coreceptor use. The effect of input copy number on population-based sequencing of the V3 loop of HIV-1 was examined through replicate deep and population-based sequencing of samples with known tropism, a heterogeneous clinical sample (624 population-based sequences and 47 deep-sequencing replicates), and a large cohort of clinical samples from phase III clinical trials of maraviroc including the MOTIVATE/A4001029 studies (n = 1,521). Proviral DNA from two independent samples from each of 101 patients from the MOTIVATE/A4001029 studies was also analyzed. Cumulative technical error occurred at a rate of 3 × 10(-4) mismatches/bp, without observed effect on inferred tropism. Increasing PCR replication increased minority species detection with an ~10% minority population detected in 18% of cases using a single replicate at a viral load of 1,072 copies/ml and in 44% of cases using three replicates. The nucleotide prevalence detected by population-based and deep sequencing were highly correlated (Spearman's ρ, 0.73), and the accuracy increased with increasing input copy number (P < 0.001). Triplicate sequencing was able to predict tropism changes in the MOTIVATE/A4001029 studies for both low (P = 0.05) and high (P = 0.02) viral loads. Sequences derived from independently extracted and processed samples of proviral DNA for the same patient were equivalent to replicates from the same extraction (P = 0.45) and had correlated position-specific scoring matrix scores (Spearman's ρ, 0.75; P << 0.001); however, concordance in tropism inference was only 83%. Input copy number and PCR replication are important factors in minority species detection in samples with significant heterogeneity.

Characterization of in vitro transcription amplification linearity and variability in the low copy number regime using External RNA Control Consortium (ERCC) spike-ins

Using spike-in controls designed to mimic mammalian mRNA species, we used the quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the performance of in vitro transcription (IVT) amplification process of small samples. We focused especially on the confidence of the transcript level measurement, which is essential for differential gene expression analyses. IVT reproduced gene expression profiles down to approximately 100 absolute input copies. However, a RT-qPCR analysis of the antisense RNA showed a systematic bias against low copy number transcripts, regardless of sequence. Experiments also showed that noise increases with decreasing copy number. First-round IVT preserved the gene expression information within a sample down to the 100 copy level, regardless of total input sample amount. However, the amplification was nonlinear under low total RNA input/long IVT conditions. Variability of the amplification increased predictably with decreasing input copy number. For the small enrichments of interest in typical differential gene expression studies (e.g., twofold changes), the bias from IVT reactions is unlikely to affect the results. In limited cases, some transcript-specific differential gene expression values will need adjustment to reflect this bias. Proper experimental design with reasonable detection limits will yield differential gene expression capability even between low copy number transcripts.

Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR

Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia were also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets.

Comparative Study Between Relative and Absolute BCR-ABL Transcript Quantification by Real Time PCR in Chronic Myeloid Leukemia

After the introduction of tirosine-kinase inhibitor in chronic myeloid leukemia (CML) treatment, the quantification of the level BCR/ABL positive cells has become essential. Since 1993, our lab has been using a sensitivity qualitative nested RT-PCR assay. Due to the need of tumor cells quantification in CML, we developed, in 2004, a quantitative test by real time PCR. The question was to define whether the relative or absolute quantification was the best strategy. To answer this question our lab team standardized both methods and compared them. The real time PCR (RQ-PCR) was performed in Applied Biosystems® 7500 plataform with TaqMan® probes towards b2a2, b3a2 BCR-ABL transcript and BCR reference gene, in a non multiplex assay. Two separate RQ-PCR reactions were prepared for the BCR-ABL standard and BCR standard. 129 peripheral blood samples of CML patients were tested by both relative and absolute methods. Quality control samples were analyzed in every RQ-PCR run to monitor assay performance: NTC, positive and negative control.Relative Quantification is based on the expression levels of a target gene (BCR-ABL) versus a reference gene (BCR). This method determines the mRNA level of BCR-ABL gene across mutiple samples and expresses it relative to the levels of an internal control. A pool of c-DNA of 30 patients with untreated CML in chronic phase was used as an internal control (N Engl J Med, 2003, oct 9, 349–15). This method does not require standards with known concentrations and we used Pfaffl mathematical model to calculate the expression of a target gene in relation to a reference gene (Nucl Acid Res. 2001; 29:2002–7). The relative expression ratio is calculated from the real time PCR efficiencies and the crossing point of an unknown sample versus a control: Ratio= E(target) ΔCt target (controlsample)/E(reference) ΔCt reference (control-sample)Absolute Quantification determines the input copy number of the BCR-ABL transcript, usually by relating the PCR signal to a standard curve. The standard curve was constructed using plasmids. Plasmids contaning a cDNA fragment of genes under analysis (b2a2, b3a2 BCR-ABL transcript and BCR gene) were prepared by PCR cloning (Branford S, Br J Haematol , 1999; 107:508–99). A 10-fold diluition series in the range of 106 to 10 copies was prepared for the BCR-ABL transcripts and BCR gene. The results were reported as a ratio of BCR-ABL/BCR % and were expressed relative to the median of BCR-ABL transcripts in the blood of 30 patients with untreated CML in chronic phase (baseline).Results: We performmed 129 blood samples of CML patients by both the relative and the absolute RQ-PCR. The results showed a positive correlation between relative and absolute values (r = 0.969 and p =0.000).Conclusion: The results of relative and absolute RQ-PCR methods were equivalent. Although the absolute method is more frequent in literature, the relative quantification presents more simply standardization, low contamination risk since it does not work with plasmids and results as safe as the absolute RQ-PCR.

Validation of the 2-????Ct Calculation as an Alternate Method of Data Analysis for Quantitative PCR of BCR-ABL P210 Transcripts

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder that is characterized by the presence of a reciprocal translocation between chromosomes 9 and 22 and results in the formation of the Philadelphia (Ph1) chromosome and is present in most of CML patients. The Ph1 chromosome forms a chimeric gene that encodes an abnormal P210 mRNA transcript in most CML patients. Surveillance for minimal residual disease by detection of BCR/ABL transcripts is currently done mostly by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Quantitation of BCR/ABL transcripts can monitor tumor load and the outcome of therapy. Absolute quantification determines the input copy number of the transcript of interest, usually by plotting the amount of PCR product onto a standard curve based on serial dilutions of the same product cloned in plasmids. Relative quantification describes the change in expression of the target gene in the patient sample relative to that of a control transcript by using the 2-DeltaDeltaCt calculation. The results of real-time RT-PCR for BCR/ABL transcripts are often analyzed by using plasmid DNA standard curves. In the present study, 79 BCR/ABL transcript-positive samples from CML patients who were being monitored for minimal residual disease by real-time quantitative RT-PCR were studied to determine whether the 2-DeltaDeltaCt approach was equivalent to the plasmid standard curve method. BCR/ABL P210 transcripts were quantitated using both the plasmid standard curve method and the 2-DeltaDeltaCt calculation. The comparison of both methods revealed a highly significant and linear correlation between the plasmid standard curve method and the 2-DeltaDeltaCt calculation (R2=0.98, P<0.0001). Furthermore, there was a reduction of preparation time, contamination risk, and reagent usage. The 2-DeltaDeltaCt calculation is a convenient alternative method to derive accurate quantitative information from real time PCR assays.

Automatic registration of intraoperative MRI for surgical navigation— a phantom study

The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2−ΔΔCT method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2−ΔΔCT method. In addition, we present the derivation and applications of two variations of the 2−ΔΔCT method that may be useful in the analysis of real-time, quantitative PCR data.

New Real-Time Reverse Transcriptase-Initiated PCR Assay with Single-Copy Sensitivity for Human Immunodeficiency Virus Type 1 RNA in Plasma

More sensitive assays for human immunodeficiency virus type 1 (HIV-1) RNA are needed to detect, quantify, and characterize persistent viremia in patients who are receiving antiretroviral therapy and whose plasma HIV-1 RNA levels are suppressed to less than 50 to 75 copies/ml. We therefore developed an internally controlled real-time reverse transcriptase-initiated PCR assay that quantifies HIV-1 RNA concentrations down to 1 copy per ml of plasma. This assay with single-copy sensitivity (the single-copy assay) generates a reproducible linear regression plot of input copy number versus threshold cycle by using HIV-1 RNA transcripts at copy numbers ranging from 1 to 10(6) per reaction mixture. The single-copy assay was compared to the ultrasensitive AMPLICOR HIV-1 MONITOR assay and a more sensitive modification of the ultrasensitive assay by repeatedly testing a low-copy-number panel containing 200 to 0.781 copies of HIV-1 RNA per ml of plasma. This comparison showed that the single-copy assay had a greater sensitivity than the other assays and was the only assay that detected HIV-1 RNA at levels as low as 0.781 copies/ml. Testing of plasma samples from 15 patients who were receiving antiretroviral therapy and who had <75 HIV-1 RNA copies/ml revealed persistent viremia in all 15 patients, with HIV-1 RNA levels ranging from 1 to 32 copies/ml (median, 13 copies/ml). The greater sensitivity of the single-copy assay should allow better characterization of persistent viremia in patients who are receiving antiretroviral therapy and whose HIV-1 RNA levels are suppressed to below the detection limits of present assays.

Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method

The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2−ΔΔCT method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2−ΔΔCT method. In addition, we present the derivation and applications of two variations of the 2−ΔΔCT method that may be useful in the analysis of real-time, quantitative PCR data.

Bacteriophage lambda DNA fragments replicate in the Paramecium macronucleus: absence of active copy number control.

We show that bacteriophage lambda DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit-length linear molecules. These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division.