Review

Authors: Himanshu Saini, Rajneesh Thakur, Rubina Gill...

CRISPR/Cas9 gene editing system is recently developed robust genome editing technology for accelerating plant breeding. Various modifications of this editing system have been established for adaptability in plant varieties as well as for its improved efficiency and portability. This review provides an in-depth look at the various strategies for synthesizing gRNAs for efficient delivery in plant cells, including chemical synthesis and transcription. It also covers traditional analytical tools and emerging developments in detection methods to analyze CRISPR/Cas9 mediated mutation in plant breeding. Additionally, the review outlines the various analytical tools which are used to detect and analyze CRISPR/Cas9 mediated mutations, such as next-generation sequencing, restriction enzyme analysis, and southern blotting. Finally, the review discusses emerging detection methods, including digital PCR and qPCR. Hence, CRISPR/Cas9 has great potential for transforming agriculture and opening avenues for new advancements in the system for gene editing in plants.

Topics: CRISPR-Cas Systems; Gene Editing; Plant Breeding; Agriculture; Blotting, Southern

PubMed: 37725519
DOI: 10.1080/21645698.2023.2256930

Authors: Joel H Wheeler, Carolyn K J Young, Matthew J Young...

A single cell can contain several thousand copies of the mitochondrial DNA genome or mtDNA. Tools for assessing mtDNA content are necessary for clinical and toxicological research, as mtDNA depletion is linked to genetic disease and drug toxicity. For instance, mtDNA depletion syndromes are typically fatal childhood disorders that are characterized by severe declines in mtDNA content in affected tissues. Mitochondrial toxicity and mtDNA depletion have also been reported in human immunodeficiency virus-infected patients treated with certain nucleoside reverse transcriptase inhibitors. Further, cell culture studies have demonstrated that exposure to oxidative stress stimulates mtDNA degradation. Here we outline a Southern blot and nonradioactive digoxigenin-labeled probe hybridization method to estimate mtDNA content in human genomic DNA samples. © 2019 by John Wiley & Sons, Inc.

Topics: Blotting, Southern; Cells, Cultured; DNA Probes; DNA Restriction Enzymes; DNA, Mitochondrial; Digoxigenin; Electrophoresis, Agar Gel; Humans; Plasmids; Staining and Labeling

PubMed: 30982231
DOI: 10.1002/cptx.75

Authors: Dan Zhou, Lei Tan, Jian Li...

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Topics: Animals; Base Sequence; Blotting, Southern; Gene Targeting; Genotype; Homologous Recombination; Mice; Mice, 129 Strain; Mouse Embryonic Stem Cells; Polymerase Chain Reaction

PubMed: 30531726
DOI: 10.3791/58467

Review

Authors: Dong-Sheng Lian, Xiang-Yuan Chen, Hua-Song Zeng...

Most hereditary diseases are incurable, but their deterioration could be delayed or stopped if diagnosed timely. It is thus imperative to explore the state-of-the-art and high-efficient diagnostic techniques for precise analysis of the symptoms or early diagnosis of pre-symptoms. Diagnostics based on clinical presentations, hard to distinguish different phenotypes of the same genotype, or different genotypes displaying similar phenotypes, are incapable of pre-warning the disease status. Molecular diagnosis is ahead of harmful phenotype exhibition. However, conventional gold-standard molecular classifications, such as karyotype analysis, Southern blotting (SB) and sequencing, suffer drawbacks like low automation, low throughput, prolonged duration, being labor intensive and high cost. Also, deficiency in flexibility and diversity is observed to accommodate the development of precise and individualized diagnostics. The aforementioned pitfalls make them unadaptable to the increasing clinical demand for detecting and interpreting numerous samples in a rapid, accurate, high-throughput and cost-effective manner. Nevertheless, capillary electrophoresis based on genetic information analysis, with advantages of automation, high speed, high throughput, high efficiency, high resolution, digitization, versatility, miniature and cost-efficiency, coupled with flexible-designed PCR strategies in sample preparation (PCR-CE), exhibit an excellent power in deciphering cryptic molecular information of superficial symptoms of genetic diseases, and can analyze in parallel a large number of samples in a single PCR-CE, thereby providing an alternative, accurate, customized and timely diagnostic tool for routine screening of clinical samples on a large scale. Thus, the present study focuses on CE-based nucleic acid analysis used for inherited disease diagnosis. Also, the limitations and challenges of this PCR-CE for diagnosing hereditary diseases are discussed.

Topics: Biomarkers; Blotting, Southern; Electrophoresis, Capillary; Genetic Diseases, Inborn; Genotype; High-Throughput Screening Assays; Humans; Nucleic Acids; Polymerase Chain Reaction; Spectrometry, Fluorescence

PubMed: 32374277
DOI: 10.1515/cclm-2020-0186

Review

Authors: Anamaria G Zavala, Amit S Kulkarni, Elizabeth A Fortunato...

This report describes the development of a novel dual color Southern protocol to visualize two distinct genomes or genic regions simultaneously on a single Southern blot. The blot is developed with IRDye-conjugated antibody (Ab) and streptavidin that recognize digoxigenin (Dig)- or biotin-labeled probes, respectively and visualized on an infrared imager. This protocol was validated by visualizing viral and host genomes of human cytomegalovirus (HCMV)-infected human fibroblasts. This technique utilizes extremely sensitive fluorescent imaging, allowing the detection of nanogram quantities of DNA, as opposed to microgram quantities needed in Southerns using radioactively labeled probes, and eliminates the inherent loss in signal after stripping and reprobing a Southern blot. The probes are labeled with non-radioactive Dig and biotin and can be stored for extended periods of time. This protocol will aid in studies of any system with two genomes, such as cells infected with numerous types of microorganisms (virus/parasites/bacteria), or studies of mitochondrial and nuclear DNA within the same cells.

Topics: Blotting, Southern; Cytomegalovirus; DNA; Fibroblasts; Fluorescent Dyes; Genome; Humans

PubMed: 24389128
DOI: 10.1016/j.jviromet.2013.12.019

Comparative Study

Authors: Daniel Nettle, Shahinaz M Gadalla, Tsung-Po Lai...

Researchers increasingly wish to test hypotheses concerning the impact of environmental or disease exposures on telomere length (TL), and they use longitudinal study designs to do so. In population studies, TL is usually measured with a quantitative polymerase chain reaction (qPCR)-based method. This method has been validated by calculating its correlation with a gold standard method such as Southern blotting (SB) in cross-sectional data sets. However, in a cross-section, the range of true variation in TL is large, and measurement error is introduced only once. In a longitudinal study, the target variation of interest is small, and measurement error is introduced at both baseline and follow-up. In this paper, we present results from a small data set (n = 20) in which leukocyte TL was measured twice 6.6 years apart by means of both qPCR and SB. The cross-sectional correlations between qPCR and SB were high at both baseline (r = 0.90) and follow-up (r = 0.85), yet their correlation for TL change was poor (r = 0.48). Moreover, the qPCR data but not the SB data showed strong signatures of measurement error. Through simulation, we show that the statistical power gain from performing a longitudinal analysis is much greater for SB than for qPCR. We discuss implications for optimal study design and analysis.

Topics: Blotting, Southern; Correlation of Data; Cross-Sectional Studies; Humans; Leukocytes; Longitudinal Studies; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Research Design; Telomere

PubMed: 33564874
DOI: 10.1093/aje/kwab025

Comparative Study

Authors: Ling-yun Gao, Guo-dong Li, Tan-jun Tong...

OBJECTIVE

To analyze and compare the performances of two telomere measurement methods (digoxigenin-labeled Southern blot and Real-time PCR) in cellular senescence research.

METHODS

Genomic DNA extracted from normal human fibroblasts (2BS) of different population doublings (PDs) was used as test samples. The Southern blot and Real-time PCR methods for telomere measurements were optimized. The specificity and sensitivity of digoxigenin detection system were analyzed by dot blot. The two methods were respectively used to measure parallel samples to analyze and compare their resolution and accuracy.

RESULTS

Digoxigenin-labeled Southern blot system could detect less than 1 μg of human genomic DNA, but the optimal sample size was around 4-5 μg when measuring telomeres. The resolution of the Southern blot method was around 150 bp while the Real-time PCR method 300-400 bp. The former could distinguish the difference of 2 PDs for 2BS cells while the latter could not distinguish the difference of less than 5 PDs. The measurement error of the repeated measurements for the Real-time PCR method was more than 10% which was bigger than that of the Southern blot method (2.5%, P<0.001).

CONCLUSION

Digoxigenin-labeled Southern blot system is fully applicable to telomere measurement. The performance of the Southern blot method is better than that of the Real-time PCR method while the latter is convenient and high-throughput. In the study of cellular senescence, the appropriate method should be selected according to specific experiment.

Topics: Blotting, Southern; Cellular Senescence; DNA; Fibroblasts; Humans; Real-Time Polymerase Chain Reaction; Telomere; Telomere Shortening

PubMed: 23591355
DOI: No ID Found

Authors: Ming-Mei Chang

Southern blot analysis is an important molecular biology technique for identifying a specific sequence in DNA samples. Although it is no longer used extensively in recent years, the steps and underlying principles of Southern blot are applicable to modern biology. High sensitivity and limited background are keys to successful Southern blots, whereas obtaining good quality and quantity of genomic DNA as starting materials and detecting a single/low copy target sequence in the genome can be challenging. To ensure student success in performing the technique for the first time, a modified "plasmid-to-plasmid" Southern blot was implemented to confirm the presence of grape nucleotide-binding site (nbs) sequences in cloned plasmids like those described previously. The plasmid DNA and a control plasmid, pSCA7 (T1-T3-W6) containing a known grape nbs sequence, were digested with restriction enzymes, followed by agarose gel electrophoresis. The DNA band corresponding to the nbs sequence of the pSCA7 (T1-T3-W6) was extracted from the gel for PCR digoxigenin (DIG) probe synthesis. At the same time, the cloned plasmid DNA and its digested DNA fragments were blotted from the gel onto nylon membranes to be hybridized with the DIG probe followed by the detection for nbs sequences. Students successfully performed Southern blots to confirm the presence of nbs sequences in their cloned plasmids and wrote up the results following the format of scientific research papers. They learned the principles and applications of Southern blot and gained hands-on experience with associated techniques.

Topics: Binding Sites; Blotting, Southern; DNA; Humans; Nucleotides; Plasmids

PubMed: 35791664
DOI: 10.1002/bmb.21642

Authors: Carmen M Martin-Ruiz, Duncan Baird, Laureline Roger...

BACKGROUND

Telomere length is a putative biomarker of ageing, morbidity and mortality. Its application is hampered by lack of widely applicable reference ranges and uncertainty regarding the present limits of measurement reproducibility within and between laboratories.

METHODS

We instigated an international collaborative study of telomere length assessment: 10 different laboratories, employing 3 different techniques [Southern blotting, single telomere length analysis (STELA) and real-time quantitative PCR (qPCR)] performed two rounds of fully blinded measurements on 10 human DNA samples per round to enable unbiased assessment of intra- and inter-batch variation between laboratories and techniques.

RESULTS

Absolute results from different laboratories differed widely and could thus not be compared directly, but rankings of relative telomere lengths were highly correlated (correlation coefficients of 0.63-0.99). Intra-technique correlations were similar for Southern blotting and qPCR and were stronger than inter-technique ones. However, inter-laboratory coefficients of variation (CVs) averaged about 10% for Southern blotting and STELA and more than 20% for qPCR. This difference was compensated for by a higher dynamic range for the qPCR method as shown by equal variance after z-scoring. Technical variation per laboratory, measured as median of intra- and inter-batch CVs, ranged from 1.4% to 9.5%, with differences between laboratories only marginally significant (P = 0.06). Gel-based and PCR-based techniques were not different in accuracy.

CONCLUSIONS

Intra- and inter-laboratory technical variation severely limits the usefulness of data pooling and excludes sharing of reference ranges between laboratories. We propose to establish a common set of physical telomere length standards to improve comparability of telomere length estimates between laboratories.

Topics: Aging; Biomarkers; Blotting, Southern; DNA; Humans; International Cooperation; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Telomere

PubMed: 25239152
DOI: 10.1093/ije/dyu191

Authors: Shannon Owens, Shangming Tang, Neil Hunter...

Homologous recombination is fundamental to sexual reproduction, facilitating accurate segregation of homologous chromosomes at the first division of meiosis, and creating novel allele combinations that fuel evolution. Following initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs), homologous pairing and DNA strand exchange form joint molecule (JM) intermediates that are ultimately resolved into crossover and noncrossover repair products. Physical monitoring of the DNA steps of meiotic recombination in Saccharomyces cerevisiae (budding yeast) cultures undergoing synchronous meiosis has provided seminal insights into the molecular basis of meiotic recombination and affords a powerful tool for dissecting the molecular roles of recombination factors. This chapter describes a suit of electrophoretic and Southern hybridization techniques used to detect and quantify the DNA intermediates of meiotic recombination at recombination hotspots in budding yeast. DSBs and recombination products (crossovers and noncrossovers) are resolved using one-dimensional electrophoresis and distinguished by restriction site polymorphisms between the parental chromosomes. Psoralen cross-linking is used to stabilize branched JMs, which are resolved from linear species by native/native two-dimensional electrophoresis. Native/denaturing two-dimensional electrophoresis is employed to determine the component DNA strands of JMs and to measure the processing of DSBs. These techniques are generally applicable to any locus where the frequency of recombination is high enough to detect intermediates by Southern hybridization.

Topics: Blotting, Southern; Electrophoresis; Genetic Techniques; Homologous Recombination; Meiosis; Saccharomyces cerevisiae

PubMed: 29523236
DOI: 10.1016/bs.mie.2017.12.005