Accurate DNA Quantification Research Articles

A nuclear DNA-based species determination and DNA quantification assay for common poultry species

DNA testing for food authentication and quality control requires sensitive species-specific quantification of nuclear DNA from complex and unknown biological sources. We have developed a multiplex assay based on TaqMan® real-time quantitative PCR (qPCR) for species-specific detection and quantification of chicken (Gallus gallus), duck (Anas platyrhynchos), and turkey (Meleagris gallopavo) nuclear DNA. The multiplex assay is able to accurately detect very low quantities of species-specific DNA from single or multispecies sample mixtures; its minimum effective quantification range is 5 to 50pg of starting DNA material. In addition to its use in food fraudulence cases, we have validated the assay using simulated forensic sample conditions to demonstrate its utility in forensic investigations. Despite treatment with potent inhibitors such as hematin and humic acid, and degradation of template DNA by DNase, the assay was still able to robustly detect and quantify DNA from each of the three poultry species in mixed samples. The efficient species determination and accurate DNA quantification will help reduce fraudulent food labeling and facilitate downstream DNA analysis for genetic identification and traceability.

Concordance Study: Methods of Quantifying Corn and Soybean Genomic DNA Intended for Real-Time Polymerase Chain Reaction Applications

Quantitative real-time polymerase chain reaction (qPCR) is a technology commonly used for the detection and quantification of genetically engineered (GE) traits in grains and oilseeds. The method involves measuring copy numbers of taxon-specific, endogenous control genes exposed to the same manipulations as trait-specific target genes. Accurate DNA quantification is essential for successful and predictable results with qPCR. A systematic study of seven different DNA quantification methods, incorporating different chemistries and different instrumentation, were evaluated on corn and soy DNA that was extracted using two distinct extraction methods. A time course study showed that corn and soy DNA was stable under typical laboratory storage conditions. CornCTAB and cornQiagen DNA extracts produced statistically similar quantification values when measured by picogreen PG(TD700), PG(Lum20/20), Hoescht(TD700), and Hoescht(Lum20/20) methods, suggesting that these methods can be used interchangeably to quantify DNA in corn samples prior to initiation of qPCR. Soy(Qiagen) provided greater stochastic measurement variability when quantification methods were compared, whereas soyCTAB had statistically significant differences when a PG method was compared to a Hoescht method of DNA quantification. Finally, agarose gel electrophoresis data revealed more pronounced degradation for Qiagen-extracted DNA compared with CTAB extracts in both corn and soy. Consequently, Ct values generated by qPCR suggested that absolute copy numbers of PCR amplifiable targets were not concordant between Qiagen and CTAB DNA extracts. Understanding measurement uncertainty from component steps used in qPCR can contribute toward harmonizing methods for the detection of GE traits in grains and oilseeds.

Development of salt-tolerance interface for an high performance liquid chromatography/inductively coupled plasma mass spectrometry system and its application to accurate quantification of DNA samples

Accurate quantification of DNA is highly important in various fields. Determination of phosphorus by ICP-MS is one of the most effective methods for accurate quantification of DNA due to the fixed stoichiometry of phosphate to this molecule. In this paper, a smart and reliable method for accurate quantification of DNA fragments and oligodeoxythymidilic acids by hyphenated HPLC/ICP-MS equipped with a highly efficient interface device is presented. The interface was constructed of a home-made capillary-attached micronebulizer and temperature-controllable cyclonic spray chamber (IsoMist). As a separation column for DNA samples, home-made methacrylate-based weak anion-exchange monolith was employed. Some parameters, which include composition of mobile phase, gradient program, inner and outer diameters of capillary, temperature of spray chamber etc., were optimized to find the best performance for separation and accurate quantification of DNA samples. The proposed system could achieve many advantages, such as total consumption for small amount sample analysis, salt-tolerance for hyphenated analysis, high accuracy and precision for quantitative analysis. Using this proposed system, the samples of 20 bp DNA ladder (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, 500 base pairs) and oligodeoxythymidilic acids (dT 12–18) were rapidly separated and accurately quantified.

Chromatin Immunoprecipitation (ChIP): Revisiting the Efficacy of Sample Preparation, Sonication, Quantification of Sheared DNA, and Analysis via PCR

The “quantitative” ChIP, a tool commonly used to study protein-DNA interactions in cells and tissue, is a difficult assay often plagued with technical error. We present, herein, the process required to merge multiple protocols into a quick, reliable and easy method and an approach to accurately quantify ChIP DNA prior to performing PCR. We demonstrate that high intensity sonication for at least 30 min is required for full cellular disruption and maximum DNA recovery because ChIP lysis buffers fail to lyse formaldehyde-fixed cells. In addition, extracting ChIP DNA with chelex-100 yields samples that are too dilute for evaluation of shearing efficiency or quantification via nanospectrophotometry. However, DNA extracted from the Mock-ChIP supernatant via the phenol-chloroform-isoamyl alcohol (PCIA) method can be used to evaluate DNA shearing efficiency and used as the standard in a fluorescence-based microplate assay. This enabled accurate quantification of DNA in chelex-extracted ChIP samples and normalization to total DNA concentration prior to performing real-time PCR (rtPCR). Thus, a quick ChIP assay that can be completed in nine bench hours over two days has been validated along with a rapid, accurate and repeatable way to quantify ChIP DNA. The resulting rtPCR data more accurately depicts treatment effects on protein-DNA interactions of interest.

Developmental Validation of Feline, Bovine, Equine, and Cervid Quantitative PCR Assays*

Accurate DNA quantification is essential for optimizing DNA testing and minimizing sample consumption. Real-time quantitative polymerase chain reaction (qPCR) assays have been published for human and canine nuclear DNA, and the need for quantifying other forensically important species was evident. Following the strategy employed for the canine qPCR assay, we developed individual assays to accurately quantify feline, bovine, equine, and cervid nuclear DNA. Each TaqMan-based assay incorporates a genus-specific probe targeting the Melanocortin-1 Receptor gene and includes a piece of synthetic DNA that acts as an internal PCR control for detecting inhibition. Developmental validations were carried out following the revised guidelines of the Scientific Working Group on DNA Analysis Methods with modifications necessary for validation of nonhuman qPCR assays. All assays demonstrated the specificity, sensitivity, stability, reproducibility, accuracy, and precision required for forensic casework. The application of these assays to animal forensic DNA analysis has both conserved laboratory resources and improved genotyping results.

Quantitative PCR methods for RNA and DNA in marine sediments: maximizing yield while overcoming inhibition

For accurate quantification of DNA and RNA from environmental samples, yield loss during nucleic acid purification has to be minimized. Quantitative PCR (qPCR) and reverse transcription (RT)-qPCR require a trade-off between maximizing yield and removing inhibitors. We compared DNA and RNA yield and suitability for quantitative SYBR Green PCR and RT-PCR using the UltraClean and PowerSoil extraction kits and a bead-beating protocol with phenol/chloroform extraction steps. Purification methods included silica-column-based procedures from the MoBio kits, RNeasy MinElute, WizardPlus miniprep columns, and an acrylamide gel extraction. DNA and RNA purification with WizardPlus and RNeasy, respectively, led to significant losses of nucleic acids and archaeal 16S rRNA or 16S rRNA gene, as measured with RiboGreen or PicoGreen, and RT-qPCR or qPCR. Extraction and purification of DNA with the MoBio DNA UltraClean and DNA PowerSoil kits also decreased the yields slightly, relative to gel purification, in all sediments, except those from the deep sea in the Gulf of Mexico. Organic matter in humic-rich sediments may bind to these silica columns, reducing their nucleic acid-loading capacity. Purification with gel extraction cleans up organic-rich sediment samples sufficiently for quantitative analysis while avoiding the yield loss associated with commonly used silica columns.

Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits

Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered.

Quantification of an Oligonucleotide Containing a Sequence Failure Product: Comparison of Isotope Dilution Mass Spectrometry with other Quantification Methods

There is an increasing demand to develop a method for accurate quantification of DNA. Because current methods such as the ultraviolet (UV) absorption-based method are only capable of relative quantification, the quantification result depends completely on the reference material. To achieve accurate quantification of DNA, we have performed isotope dilution mass spectrometry (ID MS)-based quantification of oligonucleotides. We chose a 20-mer synthetic oligonucleotide as the analyte with a longer sequence failure product. Oligonucleotides sometimes contain sequence failure products, which are difficult to remove. It is important to quantify a target product in such mixture. After evaluating the content of the sequence failure product, the analyte spiked with stable isotopically labeled deoxynucleotide monophosphates (dNMPs) was digested by enzyme to its constituent dNMPs or deoxynucleosides, and quantified by liquid chromatography-mass spectrometry. The obtained mass fractions of the 20-mer oligonucleotide showed a good agreement with the results based on phosphate analysis by inductively coupled plasma-optical emission spectrometry and ion chromatography. UV absorption, the general method for DNA quantification, resulted in underestimation. On the other hand, the mass fraction obtained by the gravimetric method was overestimated. This study shows that the ID MS method can determine the precise mass fraction of the target oligonucleotide with the sequence failure product and possesses potential as the primary method for the certification of DNA as a reference material.

Internal validation of the 7500 Real Time PCR System for use in forensic casework in Hellenic Police

According to guidelines of the quality assurance standards for the forensic DNA testing laboratories, prior to introducing an existing DNA analysis procedure, reliability of the procedure has to be demonstrated by carrying out internal validation. In order to introduce a sensitive and accurate DNA quantification method in our laboratory, a validation study of the 7500 Real Time PCR System (Applied Biosystems) using the QUANTIFILER Human DNA Quantification Kit (Applied Biosystems) was performed. Here we report the results and the experience we have gained during this internal validation study.

DNA Quantification of Whole Genome Amplified Samples for Genotyping on a Multiplexed Bead Array Platform

Whole genome amplification (WGA) permits genotyping DNA samples of limited quantity, expanding the number of samples available for genetic epidemiology studies. WGA, however, produces various nucleic acid side products that may interfere with accurate DNA quantification and further analysis. Although quality control of whole genome amplified DNA (wgaDNA) before genotyping is essential to prevent failed or poor genotyping results, little information is available to choose the best method for wgaDNA quantification. Therefore, we quantified wgaDNA from 54 buccal or poor quality blood samples by four methods: UV absorbance, PicoGreen fluorescence calibrated with lambda bacteriophage or calf thymus DNA, and absolute quantification by real-time PCR amplification of human-specific Alu Yd6. We then genotyped these wgaDNA samples and paired high-quality genomic DNA samples on a custom 384-plex Illumina Golden Gate Panel. Of the 54 paired samples, 39 gave high concordance (>99%), whereas 7 had moderate concordance (>90-99%) and 8 had poor concordance (</=90%) of wgaDNA and genomic DNA genotyping results. Quantitative PCR of Alu was the only wgaDNA quantification method to distinguish wgaDNA samples that gave high, moderate, or low concordance results (i.e., wgaDNA quantities in the high, moderate, and poor concordance groups ranged at 4.14-118.32, 0.29-2.19, and 0.01-0.27 ng/microL, respectively). Human-specific quantitative PCR is a highly useful guide for determining the suitability of wgaDNA before high-throughput single-nucleotide polymorphism analysis.

Technique for Quantitative Detection of Specific DNA Sequences Using Alternately Binding Quenching Probe Competitive Assay Combined with Loop-Mediated Isothermal Amplification

We describe a novel technique for a simple, rapid, and reliable quantitative detection of specific DNA sequences using an alternately binding quenching probe (AB-QProbe) that binds to either the gene of interest (target) or an internal standard (competitor) in combination with loop-mediated isothermal amplification (LAMP). The AB-QProbe is a singly labeled oligonucleotide bearing a fluorescent dye at the 5' end. The fluorescence intensity of the AB-QProbe reflects the ratio of the LAMP products from the target and competitor. We amplified the target and competitor by LAMP under isothermal conditions with high specificity, efficiency, and rapidity and calculated the starting quantity of the target from the fluorescence intensities at the beginning and end of LAMP. We call this technique alternately binding quenching probe competitive LAMP (ABC-LAMP). We quantified amoA, which encodes the ammonia-oxidizing enzyme in environmental bacteria, as a model target by ABC-LAMP, real-time PCR, and real-time turbidimetry of LAMP. By comparison, the accuracy of ABC-LAMP was found to be similar to that of real-time PCR. Moreover, ABC-LAMP enables the accurate quantification of DNA in the presence of DNA amplification inhibitors such as humic acid, urea, and Triton X-100 that compromise the values measured by real-time PCR and real-time turbidimetry of LAMP.

Quantitative Method for Specific Nucleic Acid Sequences Using Competitive Polymerase Chain Reaction with an Alternately Binding Probe

We have developed a simple, cost-effective, and accurate method for the quantification of specific nucleic acid sequences by the combined use of competitive PCR and a sequence-specific fluorescent probe that binds to either the gene of interest (target) or internal standard (competitor), referred to as alternately binding probe (ABProbe). In this method, the target and competitor were coamplified with the ABProbe, and then the fluorescence intensity was measured. The ratio of the target to the competitor can be calculated from the fluorescence intensity of the ABProbe using fluorescence quenching and fluorescence resonance energy transfer, that is, the starting quantity of the target is successfully calculated by end-point fluorescence measurement. Therefore, this method eliminates the complex post-PCR steps and expensive devices for real-time fluorescence measurement. We called this method alternately binding probe competitive PCR (ABC-PCR). We quantified amoA as a model target by ABC-PCR and real-time PCR. By comparison, the sensitivity, accuracy, and precision of ABC-PCR were similar to those of real-time PCR. Moreover, ABC-PCR was able to correctly quantify DNA even when PCR was inhibited by humic acid; therefore, this method will enable accurate DNA quantification for biological samples that contain PCR inhibitors.

Real-Time PCR Assay for Detection and Quantification of Hepatitis B Virus Genotypes A to G

The detection and quantification of hepatitis B virus (HBV) DNA play an important role in diagnosing and monitoring HBV infection as well as assessing therapeutic response. The great variability among HBV genotypes and the enormous range of clinical HBV DNA levels present challenges for PCR-based amplification techniques. In this study, we describe the development, evaluation, and validation of a novel real-time PCR assay designed to provide accurate quantification of DNA from all eight HBV genotypes in patient plasma specimens. A computer algorithm was used to design degenerate real-time PCR primers and probes based upon a large number (n = 340) of full-length genomic sequences including HBV genotypes A to H from Europe, Africa, Asia, and North and South America. Genotype performance was tested and confirmed using 59 genotype A to G specimens from two commercially available worldwide genotype panels. This assay has a dynamic range of at least 8 log(10) without the need for specimen dilution, good clinical intra- and interassay precision, and excellent correlation with the Bayer Diagnostics VERSANT HBV DNA 3.0 (branched DNA) assay (r = 0.93). Probit analysis determined the 95% detection level was 56 IU/ml, corresponding to 11 copies per PCR well. The high sensitivity, wide linear range, good reproducibility, and genotype inclusivity, combined with a small sample volume requirement and low cost, make this novel quantitative HBV real-time PCR assay particularly well suited for application to large clinical and epidemiological studies.

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G + C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G + C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured.

Development of the pipetting error sensor

An air volume flow rate sensor with a resolution of nano-liter per second is developed to monitor the pipetting errors of the polymerase chain reaction (PCR) mixture dispense processes for DNA quantification experiments. Based on the CMOS and post-CMOS micromachining processes, the sensor which comprises microstructures and mixed signal processor in a die size of 2 mm square is fabricated and is set within a standard 200 μL pipette tip. The microstructures of the sensor consist of a polysilicon microheater and a thermopiles array. The centered microheater encircled by temperature sensors generates the heat plume. As the sensor is positioned against the flow, the thermal plume will be pushed towards to the temperature sensors array and the temperature difference can be converted into voltage to determine the air volume flow rate through the tip. The total volume of air movement can be correlated to liquid volumetric delivery and then the pipetting error can be tracked by the non-contact method. The DNA quantification experiments with hepatitis B virus (HBV) plasmid standard samples are performed on a real-time PCR machine as the pipetting error is monitored by the proposed sensor to reject large discrepancy in mean delivery volumes. The DNA quantification results for the serial samples with the initial template concentration ranging from 10 4 to 10 8 copies/mL give high linear standard curves, and the coefficient of variation (CV) of DNA quantification experiments for the three replicates intra-assay is less than 6%. These results indicate that accurate DNA quantification and high reproducibility of experiments can be obtained by the pipetting error sensor.

Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

BackgroundReal-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available.ResultsFive commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated.ConclusionThe crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR.

Use of Green Fluorescent Protein-Expressing Aspergillus fumigatus conidia to Validate Quantitative PCR Analysis of Air Samples Collected on Filters

This study used green fluorescent protein (GFP)-expressing Aspergillus fumigatus conidia to compare quantitative PCR (qPCR) enumeration with direct epifluorescent microscopic filter counts of conidia collected on filters in a test chamber. In separate experiments this study initially compared white versus fluorescent light microscopy for counting A. fumigatus conidia, then compared fluorescent microscopy counting of corresponding filter halves, and finally compared qPCR enumeration to counting by fluorescent light microscopy. The use of GFP-expressing conidia with epifluorescent microscopy yielded significantly higher conidia counts (p = 0.026, n = 41, mean of 4.1 conidia per counting field) and 40% faster counting times when compared to conventional counting using white light microscopy. GFP-expressing conidia were aerosolized in a test chamber and collected onto filters. Filters were divided in half and GFP-expressing conidia enumerated. There was no significant difference in the average conidia count per field between corresponding filter halves (p = 0.3, n = 9 filters, mean of 7.8 conidia per counting field). Thus, one filter half could be counted optically and would provide a reliable estimate of filter loading of the corresponding half, which could then be analyzed by qPCR. Filters (n = 38) loaded with GFP conidia in the aerosol chamber were divided in half and analyzed by either fluorescent microscopy or qPCR. The estimated filter loadings ranged from 15–30,000 conidia per filter. There was a linear relationship with a nearly 1:1 ratio between qPCR and direct microscopic estimates of filter loading (y = 1.06× + 404; R 2 = 0.91) showing that the outlined qPCR analysis method is in agreement with an external reference method and is reliable for enumerating A. fumigatus conidia collected on filters. The comparative data derived using GFP-expressing conidia confirmed that qPCR provides sensitive and accurate quantification of DNA from airborne conidia collected on filters.

Theoretical and Experimental Dissection of Competitive PCR for Accurate Quantification of DNA

We frequently use competitive PCR in the plateau phase in quantifying DNA species with a small number of cells. However, the basic issues of this method are poorly understood. Here, first we analyze this method theoretically under a generalized condition that competitor and target DNA products accumulate with different amplification efficiencies. We show a theoretical reason that competitive PCR might quantify DNA more accurately during the plateau phase than during the exponential phase. Second, we demonstrate that the theoretical predictions are supported by the experimental results of β-globin gene amplification using the lysates of human diploid fibroblast WS1 cells. We also demonstrate that we can correctly quantify target DNA by keeping the starting concentration of target DNA close to a constant preset value while using a constant number of PCR cycles and by using WS1 cells as control. Finally, we show the experimental errors in routine measurements of c-myc copy number/cell in human leukemia HL-60 cells with various levels of c-myc multiplication. The number of c-myc copies/cell was determined with an error rate of less than 10%, where agarose gel bands were stained with ethidium bromide for the product quantitation.

Determining Gene Dosage

Numerous investigations (both clinical and basic) in today’s molecular biology laboratory require accurate quantification of DNA by the polymerase chain reaction (PCR). To name a few important examples, quantitative PCR has been used to quantify viral copy number, to perform gene expression studies, and to diagnose genetic diseases. In this issue of Clinical Chemistry , Poropat and Nicholson describe a quantitative PCR assay for the determination of gene dosage (i.e., the number of copies of a gene per somatic cell) of the PMP22 gene, a myelin gene involved in inherited neuropathies (1). The PMP22 gene is extremely sensitive to copy number; for example, it is duplicated in the autosomal dominant Charcot-Marie Tooth type I (CMT1A) disease and is deleted in the autosomal dominant hereditary neuropathy with liability to pressure palsies (HNPP). Because the expression of the disorders can be quite variable and CMT1A shows genetic heterogeneity, the DNA testing for these diseases establishes a secure diagnosis; it also permits genetic counseling and testing for high-risk family members. For molecular laboratories involved in genetic disorders, gene dosage determinations are clinically important and frequently performed. In addition to CMT1A and HNPP, there are many genetic disorders where the primary defect is due either to allelic deletions (e.g., Duchenne muscular dystrophy, spinal muscular atrophy, α-thalassemia, growth hormone deficiency, and familial hypercholesterolemia) or to duplications (e.g., Klinefelter syndrome and Down syndrome). Furthermore, for the determination of the carrier state of disorders such as Duchenne muscular dystrophy and spinal muscular atrophy, the accurate determination of heterozygous deletions is essential. Originally, gene dosage determinations were made from Southern blots. The protocol was laborious and required large amounts of DNA. Furthermore, optimal conditions were required; very high-quality blots were necessary, with consistent transfer and hybridization and low background. We have found that Southern autoradiographic bands >10 kb …