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Protein Expression and Purification Aug 2023The polymerase chain reaction is an extensively used technique with numerous applications in the field of biological sciences. In addition to naturally occurring DNA...
The polymerase chain reaction is an extensively used technique with numerous applications in the field of biological sciences. In addition to naturally occurring DNA polymerases with varying processivity and fidelity, genetically engineered recombinant DNA polymerases are also used in PCR. The Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA binding protein, to the polymerase domain of the Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity. Expensive commercial variants of Pfu-Sso7d are sold under various trade names. Here, we report a quick, cost and time-efficient purification protocol and an optimized buffer system for Pfu-Sso7d. We evaluated precipitation efficiencies of varying concentrations of ethanol and acetone and compared the activities of the precipitated enzyme. Although both the solvents efficiently precipitated Pfu-Sso7d, acetone showed better precipitation efficiency. Purified Pfu-Sso7d showed excellent activity in the PCR of templates with varying lengths and GC contents. We also report a buffer system that works with Pfu-Sso7d as efficiently as commercially available buffers. This quick and efficient purification scheme and buffer system will provide researchers cost-efficient access to fusion polymerase.
Topics: Acetone; DNA-Directed DNA Polymerase; Polymerase Chain Reaction
PubMed: 37156451
DOI: 10.1016/j.pep.2023.106276 -
Methods in Molecular Biology (Clifton,... 2024In the last few decades, molecular techniques and genetic modification have been used in genotype and phenotype studies of S. suis. Genomic modification of S. suis...
In the last few decades, molecular techniques and genetic modification have been used in genotype and phenotype studies of S. suis. Genomic modification of S. suis requires DNA acquisition and its stable insertion into the chromosome by allelic exchange. In this chapter, we described two techniques for the preparation of genomic constructs (cloning and overlapping extension PCR) and for DNA uptake (electroporation and transformation). The protocols are accompanied with examples. All described protocols were successful on our hands with the reference S. suis strain P1/7.
Topics: Electroporation; Cloning, Molecular; Polymerase Chain Reaction
PubMed: 38884909
DOI: 10.1007/978-1-0716-3898-9_4 -
Clinica Chimica Acta; International... Aug 2023Spinal muscular atrophy (SMA) is an autosomal recessive inherited neuromuscular condition caused by biallelic mutations in the survival of motor neuron 1 (SMN1) gene. A...
BACKGROUND AND AIMS
Spinal muscular atrophy (SMA) is an autosomal recessive inherited neuromuscular condition caused by biallelic mutations in the survival of motor neuron 1 (SMN1) gene. A homozygous deletion of the SMN1 gene accounts for approximately 95-98% of SMA patients. A highly homologous gene survival motor neuron 2 (SMN2) can partially compensate for SMN1 deletion, and its copy number is associated with disease severity. Population-based carrier screening by simultaneous quantification of SMN1 and SMN2 copy numbers is the best method to prevent SMA.
MATERIALS AND METHODS
In this study, a total of 516 samples were re-tested for the SMN1 copy number by using quantitative polymerase chain reaction (qPCR), multiplex ligation probe amplification (MLPA), droplet digital PCR (ddPCR), high-resolution melting (HRM) analysis, and PCR-based capillary electrophoresis (PCR/CE) simultaneously. Then, the performance of these methods was compared by using MLPA results as the reference.
RESULTS
The results of qPCR, ddPCR, HRM, and PCR/CE in detecting heterozygous deletion of SMN1 exon 7 and the results of ddPCR, HRM, and PCR/CE in detecting ≥2 copies of SMN1 exon7 are totally consistent with those of MLPA. The sensitivity and specificity of qPCR for detection of 2 copies of SMN1 exon 7 were 99.7% and 98.8%, respectively. The sensitivity and specificity of qPCR for detection of >2 copies of SMN1 exon 7 were 96.3% and 99.8%, respectively. Compared with the MLPA results, the sensitivity and specificity of qPCR and HRM for detection of heterozygous deletion of SMN1 exon 8 were 100% and 100%, respectively. They were 99.4% and 100%, respectively for detection of 2 copies, and 100% and 100%, respectively for detection of >2 copies. The results of PCR/CE in detecting SMN1 exon 8 were consistent with those of MLPA.
CONCLUSION
All these four methods show excellent performance in detecting heterozygous deletion of SMN1 exon 7. All PCR/CE results are totally concordant with those of MLPA. As the most cost-effective method, qPCR also shows high sensitivity and specificity in detecting SMN1. Taken together, our study provides useful information to select appropriate methods for SMA carrier screening.
Topics: Humans; Homozygote; Sequence Deletion; Muscular Atrophy, Spinal; Polymerase Chain Reaction; Exons; Survival of Motor Neuron 1 Protein
PubMed: 37479010
DOI: 10.1016/j.cca.2023.117496 -
Letters in Applied Microbiology Aug 2023Standard urine culture (SUC) is the current standard method for confirmation of a urinary tract infection (UTI). SUC identifies microorganisms in urine samples and...
Quantitative multiplex polymerase chain reaction in copies ml-1 linearly correlates with standard urine culture in colonies ml-1 for urinary tract infection (UTI) pathogens.
Standard urine culture (SUC) is the current standard method for confirmation of a urinary tract infection (UTI). SUC identifies microorganisms in urine samples and semi-quantifies these as colony-forming units (CFUs) ml-1. In contrast, quantitative multiplex polymerase chain reaction (q-MPCR) is a culture-independent assay in which the microbes are quantified by targeting genomic sequences and reported as cells ml-1, calculated from copies ml-1. Using serial dilutions within the 104-105 cells ml-1 range, the usual reporting range of SUC, this study compared the quantification results based on SUC and q-MPCR for four uropathogens with the control hemocytometer counts. The results revealed a linear relationship and a 1:1 correlation between the q-MPCR and SUC results. Additional q-MPCR quantification of 36 uropathogenic non-fastidious and fastidious bacteria and yeast indicated a reproducible linear correlation in a 1:1 manner with the control counts over a range of cell densities (103-106 cells ml-1). The results confirm that the quantifications by q-MPCR in cells ml-1 and by SUC in CFUs ml-1 are comparable and answer to the lingering question of how the results of these two methods correlate. Moreover, q-MPCR provided accurate quantification of various microorganisms over wider cell density ranges without the time required for microbial growth.
Topics: Humans; Multiplex Polymerase Chain Reaction; Sensitivity and Specificity; Urinary Tract Infections; Urinalysis; Bacteria
PubMed: 37500537
DOI: 10.1093/lambio/ovad085 -
Archives of Oral Biology Oct 2023This study aimed to standardize a quantitative polymerase chain reaction (qPCR)-based test to identify and quantify the uncultivable bacteria associated with...
OBJECTIVE
This study aimed to standardize a quantitative polymerase chain reaction (qPCR)-based test to identify and quantify the uncultivable bacteria associated with periodontitis.
METHODS
The standardization of qPCR, the curves for the quantification of Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis, and Filifactor alocis were developed by cloning the 16 S rRNA target gene fragment, using the GEMTEasy vector. The qPCRs were validated in 55 subgingival biofilm clinical samples, from different stages of periodontitis and from periodontally healthy/gingivitis individuals, which were previously evaluated by next-generation sequencing (NGS). The results obtained by the two methods were compared by the concordance of Cohen's Kappa index, and sensitivity, specificity, receiver operating characteristic (ROC) curve, and predictive values were established.
RESULTS
obtained by the two methods were compared using the concordance of Cohen's Kappa index, and sensitivity, specificity, predictive values, and ROC curves were generated. The qPCR test was standardized with efficiencies between 90% and 100% and R: 0.997-0.999. Concordance between the qPCR and NSG was moderate to F. alocis (agreement 78.2%; kappa 0.56, p < 0.05) and fair to the other microorganisms (agreement 67.27%-72.73; kappa 0.37-0.38, p < 0.05). qPCR exhibited a high sensitivity (82.2-100%) and specificity (100%) for E. brachy, E. saphenum, and F. alocis. Sensitivity was lower to D. oralis. Conversely, qPCR demonstrated higher sensitivity to E. saphenum than NSG (100 vs. 68.1).
CONCLUSIONS
The uncultivable microorganisms associated with periodontitis, D. oralis, E. brachy, E. saphenum, and F. alocis can be detected and quantified with the newly developed and validates qPCR test.
Topics: Humans; Porphyromonas gingivalis; Periodontitis; Gingivitis; Polymerase Chain Reaction
PubMed: 37419062
DOI: 10.1016/j.archoralbio.2023.105758 -
The Journal of Maternal-fetal &... Dec 2023The aim of this study was to evaluate if screening Group B colonization by intrapartum polymerase chain reaction could improve intrapartum administration of antibiotic...
OBJECTIVES
The aim of this study was to evaluate if screening Group B colonization by intrapartum polymerase chain reaction could improve intrapartum administration of antibiotic prophylaxis, compared with antepartum culture screening and analyze the sensitivity and specificity of polymerase chain reaction test.
METHODS
198 pregnant women with Group B colonization antepartum culture screening were included. When they arrived at hospital for delivery, two rectovaginal swabs were collected: for culture and polymerase chain reaction method.
RESULTS
The rate of Group B colonization antepartum detected by culture was 16.7%; at delivery was 17.2% when detected by culture and 19.7% using polymerase chain reaction method. The rate of inconclusive polymerase chain reaction tests was 0.5%. Considering intrapartum culture screening as gold standard, sensitivity and specificity of polymerase chain reaction test for intrapartum Group B colonization was 97.1% and 95.7%, respectively. The global rate of discordance between antepartum and intrapartum Group B colonization was 6.6%. The rate of women not treated with intrapartum antibiotic prophylaxis in the setting of positive intrapartum culture was significantly lower using intrapartum polymerase chain reaction test (0.5%) than with antepartum culture method (3.5%, = 0.035).
CONCLUSION
The use of intrapartum antibiotic prophylaxis can be more efficient when screening Group B colonization intrapartum by polymerase chain reaction test. Polymerase chain reaction method had a good performance in our study, with high sensitivity and specificity.
Topics: Pregnancy; Female; Humans; Pregnancy Complications, Infectious; Streptococcal Infections; Polymerase Chain Reaction; Parturition; Streptococcus agalactiae; Vagina
PubMed: 37766418
DOI: 10.1080/14767058.2023.2262078 -
Biotechnology Journal Apr 2024Single-stranded DNA (ssDNA) is the foundation of modern biology, with wide applications in gene editing, sequencing, DNA information storage, and materials science.... (Review)
Review
Single-stranded DNA (ssDNA) is the foundation of modern biology, with wide applications in gene editing, sequencing, DNA information storage, and materials science. However, synthesizing ssDNA with high efficiency, high throughput, and low error rate in vitro remains a major challenge. Various methods have been developed for ssDNA synthesis, and some significant results have been achieved. In this review, six main methods were introduced, including solid-phase oligonucleotide synthesis, terminal deoxynucleotidyl transferase-based ssDNA synthesis, reverse transcription, primer exchange reaction, asymmetric polymerase chain reaction, and rolling circle amplification. The advantages and limitations of each method were compared, as well as illustrate their representative achievements and applications. Especially, rolling circle amplification has received significant attention, including ssDNA synthesis, assembly, and application based on recent work. Finally, the future challenges and opportunities of ssDNA synthesis were summarized and discussed. Envisioning the development of new methods and significant progress will be made in the near future with the efforts of scientists around the world.
Topics: DNA, Single-Stranded; DNA; Polymerase Chain Reaction; DNA-Directed DNA Polymerase; Oligonucleotides; Nucleic Acid Amplification Techniques
PubMed: 38622795
DOI: 10.1002/biot.202400026 -
Journal of Fish Diseases Sep 2023Tilapia lake virus (TiLV) causes high mortality in farmed and wild tilapia in various countries. We developed a highly specific and sensitive droplet digital polymerase...
Tilapia lake virus (TiLV) causes high mortality in farmed and wild tilapia in various countries. We developed a highly specific and sensitive droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify TiLV. The ddPCR assay could detect the virus at a lower threshold than the reverse transcription-quantitative polymerase reaction (RT-qPCR) method, and the sensitivity of the ddPCR assay was 10-fold higher. The diagnostic sensitivity and specificity of the ddPCR assay were 100% and did not cross-react with tilapia tissues infected with Tilapia parvovirus, Infectious spleen and kidney necrosis virus, Aeromonas hydrophila, Streptococcus agalactiae, S. iniae and Francisella noatunensis. The assay reproducibility was demonstrated by a high correlation coefficient of 0.998, and the inter-assay coefficients of variability indicated that the ddPCR assay exhibited low variability within and between measurements. The detection limit of the TiLV ddPCR assay was 100 fg cDNA, which is equal to 3.3 copies of TiLV. Furthermore, the ddPCR assay could detect TiLV in mucus, water and infected tissue samples and the lowest copy number of TiLV detected in water samples by the ddPCR assay was 7.9 ± 0.99 copies/reaction The results of the clinical samples tested for TiLV revealed that the ddPCR assay had a relatively higher detection rate than the RT-qPCR method. Overall, the ddPCR method offers a highly promising approach for the absolute quantification of TiLV in carrier fish and samples from the environment with low viral concentrations.
Topics: Animals; Tilapia; Reproducibility of Results; Fish Diseases; Polymerase Chain Reaction; Viruses; Real-Time Polymerase Chain Reaction
PubMed: 37294665
DOI: 10.1111/jfd.13816 -
Cold Spring Harbor Protocols Sep 2023DNA recombineering uses phage λ Red recombination functions to promote integration of DNA fragments generated by polymerase chain reaction (PCR) into the bacterial...
DNA recombineering uses phage λ Red recombination functions to promote integration of DNA fragments generated by polymerase chain reaction (PCR) into the bacterial chromosome. The PCR primers are designed to have the last 18-22 nt anneal on either side of the donor DNA and to carry 40- to 50-nt 5' extensions homologous to the sequences flanking the chosen insertion site. The simplest application of the method results in knockout mutants of nonessential genes. Deletions can be constructed by replacing a portion or the entirety of a target gene with an antibiotic-resistance cassette. In some commonly used template plasmids, the antibiotic-resistance gene can be coamplified with a pair of flanking FRT (Flp recombinase recognition target) sites that, following insertion of the fragment into the chromosome, allow excision of the antibiotic-resistance cassette via the activity of the site-specific Flp recombinase. The excision step leaves behind a "scar" sequence comprising an FRT site and flanking primer annealing sequences. Removal of the cassette minimizes undesired perturbations on the expression of neighboring genes. Even so, polarity effects can result from the occurrence of stop codons within, or downstream of, the scar sequence. These problems can be avoided by the appropriate choice of the template and by designing primers so that the reading frame of the target gene is maintained past the deletion end point. This protocol is optimized for use with and .
Topics: Plasmids; Sequence Deletion; DNA; Polymerase Chain Reaction
PubMed: 36813480
DOI: 10.1101/pdb.prot107856 -
Molecular Aspects of Medicine Apr 2024The quantitative polymerase chain reaction (qPCR) is fundamental to molecular biology. It is not just a laboratory technique, qPCR is a bridge between research and... (Review)
Review
The quantitative polymerase chain reaction (qPCR) is fundamental to molecular biology. It is not just a laboratory technique, qPCR is a bridge between research and clinical practice. Its theoretical foundations guide the design of experiments, while its practical implications extend to diagnostics, treatment, and research advancements in the life sciences, human and veterinary medicine, agriculture, and forensics. However, the accuracy, reliability and reproducibility of qPCR data face challenges arising from various factors associated with experimental design, execution, data analysis and inadequate reporting details. Addressing these concerns, the Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines have emerged as a cohesive framework offering a standardised set of recommendations that describe the essential information required for assessing qPCR experiments. By emphasising the importance of methodological rigour, the MIQE guidelines have made a major contribution to improving the trustworthiness, consistency, and transparency of many published qPCR results. However, major challenges related to awareness, resources, and publication pressures continue to affect their consistent application.
Topics: Humans; Reproducibility of Results; Real-Time Polymerase Chain Reaction
PubMed: 38290180
DOI: 10.1016/j.mam.2024.101249