Determination of gene copy number and genotype of transgenic Arabidopsis thaliana by competitive PCR

A simple and rapid method is described for determining the integrated T-DNA copy number and the genotype in transgenic Arabidopsis thaliana by two-step competitive PCR. First, the amount of genomic DNA in the extracts, obtained from an individual A. thaliana transformant, was accurately determined by the 1st competitive PCR using a known single copy gene, 4HPPD (4-hydroxyphenylpyruvate dioxygenase), as a target. Second, the number of T-DNA copies per genome was estimated by quantifying the NPTII gene, which was involved in the T-DNA, by the 2nd competitive PCR using exactly the same amount of genomic DNA for each sample. The estimated copy number and genotype obtained by this procedure were identical to those determined by Southern blot analysis and segregation analysis.     

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.     

Method of extracting DNA from fine needle aspirates of human solid tumors for Southern blot analysis

A rapid, simple, convenient method for extracting DNA from fine needle aspiration (FNA) samples of human solid tumors for Southern blot hybridization studies is described. After the preparation of an air-dried cytologic smear, the remaining sample in the needle was rinsed directly into a test tube for DNA extraction. The extraction procedure, in which manipulation of the sample is minimized, produced sufficient DNA for Southern blot analysis within 24 hours of the FNA biopsy in the ten consecutive cases studied. The DNA bound to the nylon membranes can be washed and reexamined with a variety of probes, allowing studies of lymphoid cell lineage, oncogene amplification or tumor progression. The assessment of cellularity on the cytologic specimen at the time of FNA provided a reliable guide to the need for further passes to obtain sufficient cells for DNA hybridization; the cytologic diagnosis could also be made on the smears.     

Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for quantitative measurement of DNA methylation

Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) is a technique that can be used for rapid quantitation of methylation at individual CpG sites. Treatment of genomic DNA with sodium bisulfite is used to convert unmethylated Cytosine to Uracil while leaving 5-methylcytosine unaltered. Strand-specific PCR is performed to generate a DNA template for quantitative methylation analysis using Ms-SNuPE. SNuPE is then performed with oligonucleotide(s) designed to hybridize immediately upstream of the CpG site(s) being interrogated. Reaction products are electrophoresed on polyacrylamide gels for visualization and quantitation by phosphorimage analysis. The Ms-SNuPE technique is similar to other quantitative assays that use bisulfite treatment of genomic DNA to discriminate unmethylated from methylated Cytosines (i.e., COBRA, pyrosequencing). Ms-SNuPE can be used for high-throughput methylation analysis and rapid quantitation of Cytosine methylation suitable for a wide range of biological investigations, such as checking aberrant methylation changes during tumorigenesis, monitoring methylation changes induced by DNA methylation inhibitors or for measuring hemimethylation. Approximately two to four CpG sites can be interrogated in up to 40 samples by Ms-SNuPE in less than 5 h, after PCR amplification of the desired target sequence and preparation of PCR amplicons.     

A simple and rapid method for preparing genomic DNA from gram-positive bacteria

Molecular epidemiologic and other studies may require preparation of genomic DNA from large numbers of bacteria in sufficiently pure form for restriction endonuclease digestion, cloning, RAPD-PCR, Southern hybridization, and so on.Staphylococcus and other Gram-positive bacteria have a rigid cell wall and can be difficult to lyse. Here, a simple and rapid method for the preparation of genomic DNA from multiple samples is reported. This method produces clean DNA for use in most molecular biology methods in <90 min.     

Covalent genomic DNA modification patterns revealed by denaturing gradient gel blots

Several approaches are used to survey genomic DNA methylation patterns, including Southern blot, PCR, and microarray strategies. All of these methods are based on the use of methylation-sensitive isoschizomer restriction enzyme pairs and/or sodium bisulfite treatment of genomic DNA. They have many limitations, including PCR bias, lack of comprehensive assessment of methylated sites, labor-intensive protocols, and/or the need for expensive equipment. Since the presence of 5-methylcytosine alters the melting properties of DNA molecules, denaturing gradient gel blots (DGG blots), a gene scanning technique which detects differences in DNA fragments based on differential melting behavior, were used to examine genomic modification patterns in normal tissues. Variations in melting behavior, observed as restriction fragment melting polymorphisms (RFMPs), were detected in various tissues from single individuals in all human and mouse genes tested, suggesting the presence of widespread differential cell type-specific DNA modification. Additional DGG blot experiments comparing genomic DNA to unmethylated cloned DNA suggested that the melting variants were most likely caused by DNA methylation differences. The results suggest that the use of DGG blots can provide a comprehensive and rapid method for comparing complex in vivo DNA modification patterns in normal adult somatic cells.     

DNA extraction procedures meaningfully influence qPCR-based mtDNA copy number determination

Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA:nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively impact mtDNA copy number determination via qPCR. To test this we measured mtDNA:nDNA ratios in genomic DNA samples prepared using organic solvent (phenol–chloroform–isoamyl alcohol) extraction and two different silica-based column methods, and found mtDNA:nDNA ratio estimates were not uniform. We further evaluated whether different genomic DNA preparation methods could influence outcomes of experiments that use mtDNA:nDNA ratios as endpoints, and found the method of genomic DNA extraction can indeed alter experimental outcomes. We conclude genomic DNA sample preparation can meaningfully influence mtDNA copy number determination by qPCR.     

An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.

Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL‐)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T‐DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL‐PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T₁ progeny from 26 T₀ plants showed that at least 19% of the lines carried multiple T‐DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T‐DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided.     

Rapid diagnosis of Miller-Dieker syndrome and isolated lissencephaly sequence by the polymerase chain reaction

Summary Probe YNZ22 (D17S5) is a highly polymorphic, variable number tandem repeat (VNTR) marker previously shown to be deleted in all patients with the Miller-Dieker syndrome (MDS) but not in patients with isolated lissencephaly sequence (ILS). Primers were constructed to the unique sequence flanking the polymorphic, repetitive region of YNZ22 for amplification by the polymerase chain reaction (PCR). Analysis of 118 normal individuals revealed 12 alleles (differing in copy number of a 70-bp repeat unit) ranging in size from 168 to 938 bp. A retrospective study of eight MDS and six ILS patients was consistent with Southern blot analysis in all cases except one. In the latter, a very large allele (12 copies of the repeat unit) in a patient and her mother failed to amplify on initial attempts, but was successfully amplified by reducing the concentration of genomic DNA used in the reaction. Prospective studies on two MDS and five ILS patients were successfully performed and confirmed in all cases by Southern blot analysis. From the total sample, restriction fragment length polymorphism (RFLP) analysis was fully informative in four of ten MDS patients and showed a deletion in all four cases. Nine of eleven ILS patients were heterozygous and therefore not deleted for YNZ22. Development of primers for additional polymorphic markers in the Miller-Dieker region will lead to a rapid PCR-based diagnostic approach for all MDS and ILS patients. PCR typing of YNZ22 will also facilitate use of this marker in other applications, including genetic linkage, paternity and forensic studies, and analysis of loss of heterozygosity in tumors.     

An inverse PCR technique to rapidly isolate the flanking DNA of Dictyostelium insertion mutants

Restriction enzyme mediated integration is a widely used and effective method for insertional mutagenesis in Dictyostelium discoideum. In this method, plasmid rescue is used to clone the genomic deoxyribonucleic acid (DNA) sequences that flank the insertion site. For this to be effective, it is necessary to first find a convenient restriction enzyme site within the genomic DNA. This is a time-consuming process that requires Southern blot analysis of the mutant DNA. In addition, plasmid rescue requires transformation into highly competent Escherichia coli. Problems can arise owing to unstable genomic sequences, damage to the plasmid DNA and exogenous plasmid contamination. We have established a simple and rapid polymerase chain reaction-based technique that works for all mutants and circumvents the need for Southern blot analysis and plasmid rescue.     

Pre-PCR processing

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.     

High-throughput fluorescent screening of transgenic animals: phenotyping and haplotyping.

BACKGROUND: Methods for genotyping transgenic animals currently consist of extracting genomic DNA from blood or tissue followed by PCR or Southern blot analysis. These methods when used to screen large numbers of animals can be time consuming and expensive. Therefore, we developed a novel method that allows high-throughput screening of phenotypic changes on leukocytes, resulting from the transgenic genotype. This technique allows investigators to quickly screen a large number of animals without the need to extract DNA from each one. Moreover, since blood is collected for the initial screening, putative homozygotes can be confirmed by conventional methods using the same blood samples. METHODS: We collected blood from wild-type alphagal positive and alphagal knockout mice and probed for the presence of Galalpha(1-->3)Gal (alphagal) epitopes. Also, alloantigen specific antibodies were used to determine the haplotype of our outbred mouse colony in order to develop an inbred line. RESULTS: alphagal epitopes were detected in wild-type but not alphagal knock-out samples. To validate these results, PCR was used to demonstrate the native alphagal gene in wild-type and the pGKneo construct in alphagal knock-out mice. Furthermore, haplotypes were determined and mice divided for backcrosses. CONCLUSIONS: This screening method is useful for both preliminary screening of transgenic mice and the development of an inbred mouse colony by rapid determination of MHC I haplotype. Here, we demonstrate the use of this technique and show how it can be a valuable tool, saving time and resources in both investigator effort and animal husbandry.     

Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.     

Extraction of high-quality genomic DNA from latex-containing plants

The isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including long PCR, endonuclease restriction digestion, Southern blot analysis, and genomic library construction. Many protocols are available for the extraction of DNA from plant material. However, for latex-containing Asteraceae (Cichorioideae) species, standard protocols and commercially available kits do not produce efficient yields of high-quality amplifiable DNA. A cetyltrimethylammonium bromide protocol has been optimized for isolation of genomic DNA from latex-containing plants. Key steps in the modified protocol are the use of etiolated leaf tissue for extraction and an overnight 25 degrees C isopropanol precipitation step. The purified DNA has excellent spectral qualities, is efficiently digested by restriction endonucleases, and is suitable for long-fragment PCR amplification.     

Southern blot and dot blot hybridisation compared to PCR for the detection of human papillomavirus DNA in biopsies of the uterine cervix from women with dysplasia

The aim of this study was to compare the sensitivity of Southern blot (SB) and dot blot (DB) hybridisation with polymerase chain reaction (PCR) for the detection of HPV in cervical biopsies from samples with differing histology. One hundred and forty seven women with cervical dysplasia had biopsies performed; one sample was analyzed for HPV DNA from types 6/11, 16, and 18 by SB, DB and PCR (L1 consensus primer and type specific probes) while an adjacent sample was examined histologically. The histology of the samples was normal in 40 (27%), squamous metaplasia in 25 (17%), inflammation 2 (1%) HPV infection 24 (17%), cervical intraepithelial neoplasia (CIN) grade I in 11 (7%), CIN II in 18 (12%), CIN III in 22 (15%), while 5 (3%) had invasive cancer. The number of biopsies positive for HPV DNA from types 6/11, 16, and 18, using the different hybridisation methods was 56 (38%) by dot blot, 57 (39%) by Southern blot hybridisation and 66 (45%) by PCR. When the L1 consensus primer was used 100 (68%) specimens were positive by PCR. The sensitivity of SB and DB hybridisation, as compared with PCR (type specific probes 6/11, 16, 18) was greater in biopsies with abnormal histology (histological grades of HPV infection and greater, as a group) (sensitivity of SB 83%, DB 74%) than those with normal and metaplastic change (as a group) (sensitivity of SB 44%, DB 35%) (P < 0.005 for SB and DB) (inflammation excluded from analysis). This study demonstrated that the sensitivity of SB and DB hybridisation, relative to PCR is greater in samples with abnormal histology than in samples with normal histology.     

A new method for rapid screening of bacterial species- or subspecies-specific DNA probes

A simple assay for the rapid screening of bacterial species- or subspecies-specific DNA probes for the random cloning method is presented, involving the use of genomic DNAs as probes and recombinant plasmid DNAs containing genomic DNA digested with HindIII as targets. The optimal amount of target DNAs and the concentration of digoxigenin-labeled genomic DNA probes were 20 ng and 100 ng ml(-1) (or 10 ng and 200 ng ml(-1)), respectively. The method was applied to the development of Fusobacterium nucleatum subspecies-specific probes. Our results showed that four out of 96 probes were F. nucleatum subspecies-specific, which was confirmed by Southern blot analysis. Our results indicate that the new method can be used for the rapid screening of species- or subspecies-specific probes.     

Characterization of a new repetitive sequence in the Dictyostelium discoideum genome

A repetitive DNA sequence was isolated from a Dictyostelium discoideum genomic plasmid library of BglII-digested DNA ligated to the BamHI site in pBR322. This clone, called pBS582, hybridized to a large number of phage lambda Dictyostelium genomic clones. Southern blot analysis indicated that pBS582 DNA hybridized to many differently sized genomic DNA fragments generated by digestion with Eco RI, AvaI, or HindIII. Restriction maps of pBS582 and five genomic clones showed that the flanking regions of each of the genomic clones were different. These findings indicate that the sequence specific to pBS582 is scattered throughout the Dictyostelium genome and is reiterated approximately 100 times in the haploid genome. Northern blot analysis revealed that RNA which hybridized to pBS582 DNA was present during all stages of growth and development and did not seem to be developmentally regulated. Southern blot analysis of DNAs from other slime molds (D. giganteum, D. purpureum, and Polysphondylium violaceum) were performed to determine whether the pBS582 sequence was present in other species of slime molds. Hybridization of pBS582 was observed to DNA from the two Dictyostelium species but not to Polysphondylium. It may thus be possible to use hybridization of specific sequences as a biochemical tool to study the relatedness of different slime mold species and their molecular taxonomy.     

Transfer of foreign gene to giant freshwater prawn (Macrobrachium rosenbergii) by spermatophore-microinjection

We developed a spermatophore-microinjection (SMI) technique that allows exogenous DNA fragments to be transferred easily into the giant freshwater prawn (Macrobrachium rosenbergii), an important aquacultural shellfish and aquatic invertebrate model. From 28 to 1, 000 ng of the circular plasmid pGL, in a total volume of 1 microl, were directly microinjected into spermatophores. Fertilization and hatching of prawns created with SMI were completed in vivo. Fertilization and hatching rates in the SMI treatments did not differ from those of the untreated control group. The genomes of free swimming, SMI-created larvae (21 days after fertilization) were analyzed using PCR and Southern blot analyses. A product with a molecular mass of 680 bp was amplified. It corresponded to amplifications of pGL, and Southern blot analysis revealed that the amplified band was positive. The gene transfer rate was primarily dependent on the concentration of DNA during SMI. The higher the concentration of pGL, the higher the rate of gene transfer. PCR and Southern blot analyses detected the existence of foreign DNA in 16 of 23 samples (70%) of genomic DNA isolated from hatched larvae in the 750 ng pGL SMI treatment. SMI, described here for the first time, is the simplest and most efficient method for mass producing transgenic giant freshwater prawns.     

Real-time quantitative PCR-based system for determining transgene copy number in transgenic animals

In this paper, we describe a rapid and accurate real-time quantitative PCR-based system to determine transgene copy number in transgenic animals. We used the 2(-deltadeltaCt) method to analyze different transgenic lines without the requirement of a control sample previously determined by Southern blot analysis. To determine the transgene copy number in several mouse lines carrying a goat beta-Lactoglobulin transgene, we developed a TaqMan assay in which a goat genomic DNA sample was used as a calibrator. Moreover, we used the glucagon gene as a reference control because this gene is highly conserved between species and amplifies with the same efficiency and sensitivity in goat as in mouse. With this assay, we provide an alternative simple method to determine the transgene copy number, avoiding the traditional and tedious blotting techniques. The assay's discrimination ability from our results is of at least six copies and, similar to the limitations of the blotting techniques, the accuracy of the quantification diminishes when the transgene copy number is high.     

Large mitochondrial repeats multiplied during the polymerase chain reaction

The number of repeats in repetitive DNA like micro‐ and minisatellites is often determined by polymerase chain reaction (PCR). When we counted repeats in an array of mitochondrial repeats in the cattle tick (Boophilus microplus) we found that the number of repeats increased during PCR. Multiplication of the repeats was independent of the primers used to amplify the region, the PCR annealing temperature and the length of the PCR product. The use of PCR to determine the number of repeats in arrays needs to be reassessed. For long repeats, a subset of samples should always be analysed by Southern blot hybridization to confirm the PCR results.