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Molecular Biology Reports Apr 2021PCR Single-Strand Conformation Polymorphism is a method used to identify and detect mutations and is now well known for its many applications on living beings. This... (Review)
Review
PCR Single-Strand Conformation Polymorphism is a method used to identify and detect mutations and is now well known for its many applications on living beings. This paper will discuss the experimental details, limitations and sensitivity of the PCR Single-Strand Conformation Polymorphism method in relation to all existing literature available to us until today. Genomic DNA extraction, PCR amplification and Single-Strand Conformation Polymorphism conditions (concentration of polyacrylamide slab gel electrophoresis, dissociation treatment of double- stranded DNA) and comparison with PCR Restriction Fragment Length Polymorphism are presented. Since its discovery in 1989, there have been many variations, innovations, and modifications of the method, which makes it very easy, safe, fast and for this reason widely applied in clinical diagnostic, forensic medicine, biochemical, veterinary, microbiological, food and environmental laboratories. One of the possible applications of the method is the diagnosis and identification of mutations in new strains of coronaviruses, because science needs more tools to tackle the problem of this pandemic. The PCR Single-Strand Conformation Polymorphism method can be applied in many cases provided that control samples are available and the required conditions of the method are achieved.
Topics: Animals; Coronavirus; Humans; Molecular Typing; Pathology, Molecular; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single-Stranded Conformational; Sequence Analysis
PubMed: 33893925
DOI: 10.1007/s11033-021-06349-2 -
Journal of Clinical Microbiology Oct 2010The reference standard methods for drug susceptibility testing of Mycobacterium tuberculosis, such as culture on Lowenstein-Jensen or Middlebrook 7H10/11 medium, are... (Meta-Analysis)
Meta-Analysis Review
The reference standard methods for drug susceptibility testing of Mycobacterium tuberculosis, such as culture on Lowenstein-Jensen or Middlebrook 7H10/11 medium, are very slow to give results; and due to the emergence of multidrug-resistant M. tuberculosis and extensively drug-resistant M. tuberculosis, there is an urgent demand for new, rapid, and accurate drug susceptibility testing methods. PCR-single-strand conformational polymorphism (PCR-SSCP) analysis has been proposed as a rapid method for the detection of resistance to rifampin, but its accuracy has not been systematically evaluated. We performed a systematic review and meta-analysis to evaluate the accuracy of PCR-SSCP analysis for the detection of rifampin-resistant tuberculosis. We searched the Medline, Embase, Web of Science, BIOSIS, and LILACS databases and contacted authors if additional information was required. Ten studies met our inclusion criteria for rifampin resistance detection. We applied the summary receiver operating characteristic (SROC) curve to perform the meta-analysis and to summarize diagnostic accuracy. The sensitivity of PCR-SSCP analysis for the rapid detection of rifampin-resistant tuberculosis was 0.79 (95% confidence interval [CI], 0.75 to 0.82), the specificity was 0.96 (95% CI, 0.94 to 0.98), the positive likelihood ratio was 16.10 (95% CI, 5.87 to 44.13), the negative likelihood ratio was 0.20 (95% CI, 0.10 to 0.40), and the diagnostic odds ratio was 100.93 (95% CI, 31.95 to 318.83). PCR-SSCP analysis is a sensitive and specific test for the rapid detection of rifampin-resistant M. tuberculosis. Additional studies in countries with a high prevalence of multidrug-resistant M. tuberculosis and also cost-effectiveness analysis are required in order to obtain a complete picture on the utility of this method for rapid drug resistance detection in M. tuberculosis.
Topics: Antitubercular Agents; DNA, Bacterial; Drug Resistance, Bacterial; Humans; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Rifampin; Sensitivity and Specificity; Time Factors; Tuberculosis
PubMed: 20668134
DOI: 10.1128/JCM.00960-10 -
Gene Jun 2014WFIKKN2 may play a role in the regulation of muscle growth and development through its interaction with growth and differentiation factor 8 (GDF8) and growth and...
WFIKKN2 may play a role in the regulation of muscle growth and development through its interaction with growth and differentiation factor 8 (GDF8) and growth and differentiation factor 11 (GDF11), but to date research into the function of the protein has been focused on mice, even though the WFIKKN2 gene (WFIKKN2) was first identified in humans in 2001. In this study two regions (intron 1 and the 3' UTR) of ovine WFIKKN2 were investigated, using Polymerase Chain Reaction-Single Stranded Conformational Polymorphism (PCR-SSCP). Two different PCR-SSCP patterns, representing two unique DNA sequences (designated a and b) were detected in a 399-bp amplicon derived from the 3' UTR, with sequence analysis revealing one single nucleotide polymorphism (SNP). In a 421-bp amplicon from intron 1, five different PCR-SSCP patterns (designated A-E) were observed and twelve SNPs were detected. Either one or two different sequences were detected in individual sheep and all the sequences identified shared homology with the WFIKKN2 sequences from cattle and other animal species, suggesting that these sequences represent variants of the ovine WFIKKN2 gene. In intron 1 of 487 sheep from eight breeds, variants B and C were the most common, followed by A, D and E. These results indicate that ovine WFIKKN2 is polymorphic and suggest that further analysis is required to see if variation in the gene is associated with variation in growth and muscle traits in sheep.
Topics: Alleles; Animals; Breeding; Carrier Proteins; Gene Frequency; Genetic Linkage; Phylogeny; Polymorphism, Single Nucleotide; Polymorphism, Single-Stranded Conformational; Proteins; Sheep
PubMed: 24704001
DOI: 10.1016/j.gene.2014.03.062 -
Journal of Genetics Sep 2016β-lactoglobulin (β-LG) gene is suggested as a functional candidate gene for milk yield and milk composition. β-LG polymorphism has been reported to be associated with...
β-lactoglobulin (β-LG) gene is suggested as a functional candidate gene for milk yield and milk composition. β-LG polymorphism has been reported to be associated with milk yield in cows, sheep and Indian goats. This study was performed to identify SNPs in exon 7 of β-LG gene and their association with milk traits in Iranian local Mahabadi goats using polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) and PCR-sequencing. Three SSCP patterns were observed with frequencies 0.678, 0.096 and 0.226, respectively. Subsequently, after sequencing each unique pattern nine novel mutations were identified. These mutations include: T InDel at nucleotide position 93 and substitutions T/C, T/G, T/C, G/T, T/G,T/C, G/A and A/T at nucleotide positions 99, 124, 126, 134, 147, 156, 176 and 177, respectively. Of these, seven mutations were same among the genotypic patterns while differences were related to T deletion and insertion (-/T) at nucleotide position 93 with frequencies 0.22 and 0.78 in the presence and absence of T allele, respectively; and substitution (A/T) at nucleotide position 177 with frequencies 0.16 and 0.84 for A and T alleles, respectively. Milk traits including milk production (gr), milk fat and protein (%) were also measured. These findings demonstrated that β-LG gene had a significant effect on milk protein percentage (P < 0.05), but had no significant effect on milk production and milk fat percentage.
Topics: Alleles; Amino Acid Sequence; Animals; Female; Genotype; Goats; INDEL Mutation; Lactation; Lactoglobulins; Lipids; Milk; Milk Proteins; Point Mutation; Polymorphism, Single Nucleotide; Polymorphism, Single-Stranded Conformational; Quantitative Trait, Heritable; Sequence Alignment
PubMed: 27659319
DOI: 10.1007/s12041-016-0662-x -
Clinical Microbiology Reviews Oct 1999The development over the past two decades of molecular methods for manipulation of RNA and DNA has afforded molecular virologists the ability to study viral genomes in... (Comparative Study)
Comparative Study Review
The development over the past two decades of molecular methods for manipulation of RNA and DNA has afforded molecular virologists the ability to study viral genomes in detail that has heretofore not been possible. There are many molecular techniques now available for typing and subtyping of viruses. The available methods include restriction fragment length polymorphism analysis, Southern blot analysis, oligonucleotide fingerprint analysis, reverse hybridization, DNA enzyme immunoassay, RNase protection analysis, single-strand conformation polymorphism analysis, heteroduplex mobility assay, nucleotide sequencing, and genome segment length polymorphism analysis. The methods have certain advantages and disadvantages which should be considered in their application to specific viruses or for specific purposes. These techniques are likely to become more widely used in the future for epidemiologic studies and for investigations into the pathophysiology of virus infections.
Topics: Blotting, Southern; Cytomegalovirus; DNA Fingerprinting; Genome, Viral; HIV; Polymorphism, Restriction Fragment Length; Polymorphism, Single-Stranded Conformational; Reverse Transcriptase Polymerase Chain Reaction; Viruses
PubMed: 10515905
DOI: 10.1128/CMR.12.4.612 -
Blood Feb 2000The ABO blood group is clinically the most important blood group system. Elucidation of the molecular basis of the ABO polymorphism allows genotype determination without... (Comparative Study)
Comparative Study
The ABO blood group is clinically the most important blood group system. Elucidation of the molecular basis of the ABO polymorphism allows genotype determination without family studies. Described here is a new method based on the simultaneous amplification by polymerase chain reaction (PCR) of 3 fragments from exon 6, and 5' and 3' ends of exon 7 of the ABO gene, followed by single-strand conformation polymorphism (SSCP) analysis. This multiplex PCR-SSCP protocol allows the well-established base changes at 9 nucleotide positions 261, 297, 467, 526, 646, 657, 681, 1059, and 1096 to be assayed simultaneously so that 7 common alleles (A(1), A(1v), A(2), B, O(1), O(1v), and O(2)) can be distinguished in a single-tube single-lane format. Each allele was characterized by a set of 3 haplotype-specific SSCP patterns. Chinese (n = 125) and white European (n = 98) samples were analyzed, and their genotypes were found consistent with the serologic phenotypes or could be deduced unambiguously. Fifteen samples (2 Chinese and 13 white European) were each found carrying at least 1 rare allele. Most of these alleles were new and some might be generated by intragenic recombination. This technique is the simplest, quickest, and most informative method reported to date and also readily identifies new alleles. (Blood. 2000;95:1487-1492)
Topics: ABO Blood-Group System; Alleles; Asian People; Base Sequence; China; Europe; Exons; Genotype; Humans; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; White People
PubMed: 10666229
DOI: No ID Found -
Journal of Korean Medical Science Aug 1998We immunohistochemically investigated Epstein-Barr virus (EBV)-positive and negative 31 malignant lymphomas (MLs) for p53 protein using a monoclonal antibody which is...
We immunohistochemically investigated Epstein-Barr virus (EBV)-positive and negative 31 malignant lymphomas (MLs) for p53 protein using a monoclonal antibody which is expressed on a wild type and mutant human p53 protein. We evaluated the presence of mutations in exons 5 to 8 of the p53 gene using single-strand confirmation polymorphism analysis. Overexpression of p53 was detected in 13 out of 31 cases (41.9%) of MLs. However, we have documented the presence of structural alterations of the p53 gene in six cases of MLs. The presence of EBV infection in MLs was statistically unrelated to p53 protein overexpression. Excellent correlation was found between p53 immunoreactivity and histologic types of MLs. Even though the reason for discrepancy between p53 gene mutation and p53 protein overexpression remains unclear, p53 protein overexpression may be involved in the process of malignant transformation regardless of EBV infection in MLs.
Topics: Adolescent; Adult; Blotting, Southern; Child, Preschool; Female; Humans; Immunoenzyme Techniques; In Situ Hybridization; Lymphoma; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Staining and Labeling; Tumor Suppressor Protein p53
PubMed: 9741539
DOI: 10.3346/jkms.1998.13.4.361 -
Annals of Agricultural and... Jun 2021, a coccidian protozoan species, has been recently found to cause diarrhea in all age groups in immunocompetent and immunocompromised individuals in most regions of the...
INTRODUCTION AND OBJECTIVE
, a coccidian protozoan species, has been recently found to cause diarrhea in all age groups in immunocompetent and immunocompromised individuals in most regions of the world. This study aimed to conduct the molecular detection of and to determine the genetic diversity of the 18S ribosomal RNA (rRNA) gene sequence of isolated from individuals living in different provinces in Turkey by using PCR-single-strand conformation polymorphism (SSCP).
MATERIAL AND METHODS
A total of 22 subjects were included in the study. Fourteen of the subjects were female and eight were male, with ages ranging between 7-65 years. Stool specimens were examined using wet mount and modified acid-fast staining methods, which revealed the presence of oocysts in the samples. The 18S rRNA ITS-1 Ccits37f-GCTTGCTATGTTTTAGCATGTGG and Ccits501r-GCACAATGAATGCACACACA gene regions were used as primers. The PCR products were analyzed by agarose gel electrophoresis and visualized on a UV transilluminator. For the SSCP, the PCR products were denatured with formamide, run for 16 h in 6% (49:1) polyacrylamide gel, and then imaged with silver staining.
RESULTS
SSCP assay was performed given that the DNA strands demonstrated different folds; the DNA strands contain different nucleotides based on the PCR-SSCP results for the Cyclospora strains collected in 4 provinces. Moreover, 3 different band profiles were observed in the investigated samples. A slight mutation difference was observed among the strains collected.
CONCLUSIONS
Further comprehensive studies involving more -positive specimens and utilizing different mutation screening methods are warranted to demonstrate mutation differences in Cyclopora strains in Turkey.
Topics: Adolescent; Adult; Aged; Child; Cyclospora; Cyclosporiasis; DNA, Protozoan; Feces; Female; Humans; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; RNA, Ribosomal, 18S; Turkey; Young Adult
PubMed: 34184509
DOI: 10.26444/aaem/136116 -
Asian Pacific Journal of Cancer... Nov 2021A few researches evaluated the association of polymorphisms at SERPINA5 and fat mass and obesity-associated protein (FTO) genes with papillary thyroid cancer (PTC)...
BACKGROUND
A few researches evaluated the association of polymorphisms at SERPINA5 and fat mass and obesity-associated protein (FTO) genes with papillary thyroid cancer (PTC) globally. Here, we examined the presence of genetic variations within coding exon 3 of SERPINA5 gene and FTO rs9939609 polymorphism in Iranian PTC patients.
METHODS
A total of 122 patients (42 cases for SERPINA5 and 80 cases for FTO gene) and 120 healthy subjects (40 subjects or SERPINA5 and 80 subjects for FTO gene) were recruited. The genetic variation within coding exon 3 of SERPINA5 gene was evaluated by reaction-single-strand conformation polymorphism (PCR-SSCP) and FTO rs9939609 polymorphism was evaluated by RFLP-PCR assay.
RESULTS
The PCR-SSCP technique detected two rs6115G>A and rs6112T>C genetic variations within coding exon 3 of SERPINA5 gene and approved also by direct sequencing. For rs6112T>C polymorphism seven patients was heterozygous and for rs6115G>A seven PTC patients were heterozygous and two patients were homozygous.
CONCLUSION
This study indicated that SERPINA5 rs6115G>A and rs6112T>C polymorphisms might be a novel susceptibility locus for PTC in Iranian patients. However, our findings do not support an association between FTO rs9939609 polymorphism and PTC risk.
Topics: Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Case-Control Studies; Exons; Female; Humans; Iran; Male; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Protein C Inhibitor; Thyroid Cancer, Papillary; Thyroid Neoplasms
PubMed: 34837923
DOI: 10.31557/APJCP.2021.22.11.3641 -
Pathology Oncology Research : POR Oct 2016Frameshift mutation of genes containing mononucleotide repeats is a feature of gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). In the...
Frameshift mutation of genes containing mononucleotide repeats is a feature of gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). In the public genome database, we found that human HSPA4 gene encoding a heats hock protein 70 protein (HSP70-4) and MED13 gene had mononucleotide repeats in the coding sequences that could be targets for frameshift mutation in cancers with MSI. HSP70-4 is a member of HSP70 that is known to play a role in cell survival. MED13 is a member of MED genome-wide transcription regulators that function as a regulator for diverse biological processes. In this study, we analyzed the mutations in 79 GCs and 124 CRCs including high MSI (MSI-H) and microsatellite stable/low MSI (MSS/MSI-L) cases by single-strand conformation polymorphism analysis and DNA sequencing. We found frameshift mutations of HSPA4 gene in two cancers (one GC and one CRC) and MED13 gene in the other two cancers (one GC and one CRC). The frameshift mutations were deletions of one base (c.2396delA (p.Asn799MetfsX50)) in HSPA4 and (c.2175delA (p.Lys725AsnfsX4)) in MED13. Each of HSPA4 and MED13 mutations were detected in GC with MSI-H (1/34: 2.9 %) and CRC with MSI-H (1/79: 1.3 %), but not in those with MSS. Our data show that unconventional HSPA4 and MED13 genes harbored frameshift mutations in GC and CRC with MSI. These mutations might possibly inactivate their functions and could be a feature of GC and CRC with MSI-H.
Topics: Colorectal Neoplasms; DNA, Neoplasm; Frameshift Mutation; HSP110 Heat-Shock Proteins; Humans; Mediator Complex; Microsatellite Instability; Polymorphism, Single-Stranded Conformational; Stomach Neoplasms
PubMed: 27129500
DOI: 10.1007/s12253-016-0070-9