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International Journal of Molecular... Jul 2023DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an...
DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an increasing interest in developing a convenient method for preparing DNA polymerase for routine use in laboratories. We developed a method using () that expresses thermostable DNA polymerase directly in the PCR without purification. The gene was transformed into and expressed. After overnight incubation and washing, -expressing DNA polymerase (EcoliTaq) was used as the DNA polymerase without purification. EcoliTaq showed activity comparable to that of commercial DNA polymerase and remained stable for 3 months. With a high-pH buffer containing 2% Tween 20 and 0.4 M trehalose, EcoliTaq facilitated direct PCR amplification from anticoagulated whole blood samples. EcoliTaq exhibited good performance in allele-specific PCR using both purified DNA and whole blood samples. Furthermore, it proved to be useful as a DNA polymerase in hot-start PCR by effectively minimizing non-specific amplification. We developed a simple and cost-effective direct and hot-start PCR method in which EcoliTaq was used directly as a PCR enzyme, thus eliminating the laborious and time-consuming steps of polymerase purification.
Topics: Taq Polymerase; Escherichia coli; Polymerase Chain Reaction; DNA; DNA Replication
PubMed: 37511160
DOI: 10.3390/ijms241411405 -
Nature Communications Aug 2023DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases....
DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases. Analysis of DNA methylation patterns has until now been dependent on either a chemical or an enzymatic pre-treatment, which are both time consuming procedures and potentially biased due to incomplete treatment. We present a qPCR technology, EpiDirect®, that allows for direct PCR quantification of DNA methylations using untreated DNA. EpiDirect® is based on the ability of Intercalating Nucleic Acids (INA®) to differentiate between methylated and unmethylated cytosines in a special primer design. With this technology, we develop an assay to analyze the methylation status of a region of the MGMT promoter used in treatment selection and prognosis of glioblastoma patients. We compare the assay to two bisulfite-relying, methyl-specific PCR assays in a study involving 42 brain tumor FFPE samples, revealing high sensitivity, specificity, and the clinical utility of the method.
Topics: Polymerase Chain Reaction; DNA; DNA Methylation; Temperature; Oligonucleotides; CpG Islands
PubMed: 37620381
DOI: 10.1038/s41467-023-40873-y -
Cold Spring Harbor Protocols Jan 2024A simple method to determine the genetic sex of a mouse is to amplify DNA from a male-specific gene by polymerase chain reaction (PCR). This protocol is used to detect...
A simple method to determine the genetic sex of a mouse is to amplify DNA from a male-specific gene by polymerase chain reaction (PCR). This protocol is used to detect the Y-chromosome-specific gene in tissue lysates of tail tip or ear punch samples.
Topics: Mice; Male; Animals; Genotype; Y Chromosome; Polymerase Chain Reaction; DNA
PubMed: 37932078
DOI: 10.1101/pdb.prot108062 -
Biosensors Apr 2024RNA is an important information and functional molecule. It can respond to the regulation of life processes and is also a key molecule in gene expression and regulation.... (Review)
Review
RNA is an important information and functional molecule. It can respond to the regulation of life processes and is also a key molecule in gene expression and regulation. Therefore, RNA detection technology has been widely used in many fields, especially in disease diagnosis, medical research, genetic engineering and other fields. However, the current RT-qPCR for RNA detection is complex, costly and requires the support of professional technicians, resulting in it not having great potential for rapid application in the field. PCR-free techniques are the most attractive alternative. They are a low-cost, simple operation method and do not require the support of large instruments, providing a new concept for the development of new RNA detection methods. This article reviews current PCR-free methods, overviews reported RNA biosensors based on electrochemistry, SPR, microfluidics, nanomaterials and CRISPR, and discusses their challenges and future research prospects in RNA detection.
Topics: Biosensing Techniques; RNA; Humans; Electrochemical Techniques; Polymerase Chain Reaction; Nanostructures; Surface Plasmon Resonance; Microfluidics
PubMed: 38667193
DOI: 10.3390/bios14040200 -
Canadian Journal of Microbiology Nov 2023is an opportunistic pathogen known for causing hospital-acquired infections. The natural habitat includes soil, water, sewage, and drains, but it is also detected in...
is an opportunistic pathogen known for causing hospital-acquired infections. The natural habitat includes soil, water, sewage, and drains, but it is also detected in infected individuals' blood, pus, and respiratory pathways. Due to its resilient nature, it is known to be a causative agent for outbreaks. Therefore, it is crucial to understand the genetic similarity between clinical and environmental isolates. The study aimed to find the genetic relationships between clinical and environmental isolates using PCR-based typing methods such as enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), random amplified polymorphic DNA (RAPD), and repetitive extragenic palindromic sequence-based PCR (Rep-PCR). Additionally, outer membrane protein (OMP) and whole cell protein (WCP) profiles were also used. The PCR-based methods, ERIC-PCR and Rep-PCR, showed decreased genetic similarity between clinical and environmental isolates (66% and 58%, respectively). However, RAPD showed relatively higher genetic similarity (91%). The OMP and WCP profiles showed varied banding patterns between the clinical and environmental isolates in the 29-43 kDa region. The PCR-based methods proved to be a reliable and reproducible technique. The OMP and WCP profiles, though not as discriminatory as the molecular typing methods, could help identify the most and least commonly occurring protein bands and thus help in typing clinical and environmental isolates.
Topics: Humans; Random Amplified Polymorphic DNA Technique; Acinetobacter baumannii; DNA Fingerprinting; Bacterial Typing Techniques; DNA, Bacterial; Polymerase Chain Reaction
PubMed: 37364377
DOI: 10.1139/cjm-2023-0010 -
Analytical Chemistry Nov 2023We demonstrate a rapid and sensitive method for DNA detection without the need for fluorescence. This is based on carbon-coated magnetic iron (Fe) microparticles with a...
We demonstrate a rapid and sensitive method for DNA detection without the need for fluorescence. This is based on carbon-coated magnetic iron (Fe) microparticles with a covalent surface attachment of DNA. We show that these magnetic microparticles can capture complementary DNA. Significantly, the DNA covalent surface bonds are robust to high temperatures and can be included in a sample during polymerase chain reaction (PCR). This method is employed for the detection of targeted DNA sequences (40-50 bp). Hybridization probes on the surface of the magnetically susceptible Fe microparticle recognize the target DNA sequence-specifically. The double-stranded DNA (dsDNA) microparticles are then quickly captured with a magnet from the sample matrix. This foregoes postpurification processes, such as electrophoresis, which make our technique time- and cost-effective. Captured dsDNA can be detected with intercalating dyes such as ethidium bromide through a loss in the UV absorption signal with a limit of detection (LOD) of 24 nM within 15 min. Likewise, surface-bound DNA can act as a primer in PCR to decrease the LOD to 5 pM within 2 h. This is the first instance of a nucleotide-modified magnetically susceptible carbon substrate that is PCR-compatible. Besides DNA capture, this strategy can eventually be applied to sequence-specific nucleic acid purification and enrichment, PCR cleanup, and single-strand generation. The DNA-coated particles are stable under PCR conditions (unlike commonly used polystyrene or gold particles).
Topics: Carbon; DNA; Nucleic Acid Hybridization; Ethidium; Polymerase Chain Reaction; Biosensing Techniques
PubMed: 37904495
DOI: 10.1021/acs.analchem.3c02978 -
Clinical Infectious Diseases : An... Jul 2023A healthy young man first diagnosed with mpox in May 2022 presented again in November 2022 with anal proctitis and a positive polymerase chain reaction on a rectal swab...
A healthy young man first diagnosed with mpox in May 2022 presented again in November 2022 with anal proctitis and a positive polymerase chain reaction on a rectal swab for Monkeypox virus after a recent trip to Brazil, where he engaged in condomless sexual intercourse with multiple male partners.
Topics: Humans; Male; Mpox (monkeypox); Reinfection; Brazil; Monkeypox virus; Polymerase Chain Reaction
PubMed: 36905148
DOI: 10.1093/cid/ciad147 -
Journal of Applied Microbiology Nov 2023Swine respiratory disease (SRD) is a major disease complex in pigs that causes severe economic losses. SRD is associated with several intrinsic and extrinsic factors...
AIMS
Swine respiratory disease (SRD) is a major disease complex in pigs that causes severe economic losses. SRD is associated with several intrinsic and extrinsic factors such as host health status, viruses, bacteria, and environmental factors. Particularly, it is known that many pathogens are associated with SRD to date, but most of the test to detect those pathogens can be normally investigated only one pathogen while taking time and labor. Therefore, it is desirable to develop rapidly and efficiently detectable methods those pathogens to minimize the damage caused by SRD.
METHODS AND RESULTS
We designed a multiplex real-time RT-PCR (RT-qPCR) system to diagnose simultaneously 16 pathogens, including nine viruses and seven bacteria associated with SRD, on the basis of single qPCR and RT-qPCR assays reported in previous studies. Multiplex RT-qPCR system we designed had the same ability to single RT-qPCR without significant differences in detection sensitivity for all target pathogens at minimum to maximum genomic levels. Moreover, the primers and probes used in this system had highly specificity because the sets had not been detected pathogens other than the target and its taxonomically related pathogens. Furthermore, our data demonstrated that this system would be useful to detect a causative pathogen in the diagnosis using oral fluid from healthy pigs and lung tissue from pigs with respiratory disorders collected in the field.
CONCLUSIONS
The rapid detection of infected animals from the herd using our system will contribute to infection control and prompt treatment in the field.
Topics: Animals; Swine; Reverse Transcriptase Polymerase Chain Reaction; Swine Diseases; Viruses; Lung; Multiplex Polymerase Chain Reaction; Bacteria
PubMed: 37951290
DOI: 10.1093/jambio/lxad263 -
Forensic Science International Aug 2023Since its inception, DNA typing technology has been practiced as a robust tool in criminal investigations. Experts usually utilize STR profiles to identify and... (Review)
Review
Since its inception, DNA typing technology has been practiced as a robust tool in criminal investigations. Experts usually utilize STR profiles to identify and individualize the suspect. However, mtDNA and Y STR analyses are also considered in some sample-limiting conditions. Based on DNA profiles thus generated, forensic scientists often opine the results as Inclusion, exclusion, and inconclusive. Inclusion and exclusion were defined as concordant results; the inconclusive opinions create problems in conferring justice in a trial- since nothing concrete can be interpreted from the profile generated. The presence of inhibitor molecules in the sample is the primary factor behind these indefinite results. Recently, researchers have been emphasizing studying the sources of PCR inhibitors and their mechanism of inhibition. Furthermore, several mitigation strategies- to facilitate the DNA amplification reaction -have now found their place in the routine DNA typing assays with compromised biological samples. The present review paper attempts to provide a comprehensive review of PCR inhibitors, their source, mechanism of inhibition, and ways to mitigate their effect using PCR facilitators.
Topics: Microsatellite Repeats; Polymerase Chain Reaction; Nucleic Acid Amplification Techniques; DNA Fingerprinting; DNA, Mitochondrial
PubMed: 37399774
DOI: 10.1016/j.forsciint.2023.111773 -
Birth Defects Research Apr 2024Abortion and fetal death are common in fetuses with holoprosencephaly, so genetic examinations often have to be made in a post-mortem setting. The efficiency of the...
BACKGROUND
Abortion and fetal death are common in fetuses with holoprosencephaly, so genetic examinations often have to be made in a post-mortem setting. The efficiency of the conventional karyotyping using cultured fibroblasts in these situations is limited due to frequent culture failure. In the current study, archived cases of holoprosencephaly, where post-mortem genetic evaluation was requested and sufficient frozen material was available, were reevaluated using the quantitative fluorescence polymerase chain reaction (QF-PCR) technique.
METHODS
Testing for aneuploidies of chromosomes 13, 15, 16, 18, 21, 22, X, and Y with the QF-PCR technique was carried out on DNA isolated from archived frozen chorionic villi in seven cases of holoprosencephaly.
RESULTS
QF-PCR was successful in all seven cases. Two cases of trisomy 13, two cases of triploidy, and one case of trisomy 18 was found meaning a 71% diagnostic yield. The success rate of QF-PCR (100%, 7/7) was superior compared to conventional karyotyping (43%, 3/7).
CONCLUSIONS
Rapid aneuploidy testing using the QF-PCR technique is a simple, reliable, time- and cost-effective method sufficient to conclude the etiologic investigation in the majority of holoprosencephaly cases post-mortem.
Topics: Pregnancy; Female; Humans; Holoprosencephaly; Prenatal Diagnosis; Aneuploidy; Polymerase Chain Reaction; Karyotyping
PubMed: 38632851
DOI: 10.1002/bdr2.2342