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Journal of Visualized Experiments : JoVE Jul 2021In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and...
In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and robust method for genotyping individuals by Variable Number of Tandem Repeat (VNTR) analysis in the context of undergraduate laboratory classes. The human D1S80 VNTR locus is used in this protocol as a highly polymorphic marker based on variation in the number of repetitive sequences. This simple protocol conveys useful information for teachers and the implementation of DNA fingerprinting in practical laboratory classes. In the presented laboratory exercise, DNA extraction followed by PCR amplification is used to determine genetic variation at the D1S80 VNTR locus. Differences in the fragment size of PCR products are visualized by agarose gel electrophoresis. The fragment sizes and repeat numbers are calculated based on a linear regression of the size and migration distance of a DNA size standard. Following this guide, students should be able to: • Harvest and extract DNA from buccal mucosa epithelial cells • Perform a PCR experiment and understand the function of various reaction components • Analyze the amplicons by agarose gel electrophoresis and interpret the results • Understand the use of VNTRs in DNA fingerprinting and its application in biological sciences.
Topics: Alleles; DNA Fingerprinting; Humans; Laboratories; Minisatellite Repeats; Paternity; Phylogeny
PubMed: 34338668
DOI: 10.3791/62305 -
International Journal of Surgery... Jun 2018Endothelial nitric oxide synthase (eNOS) polymorphisms have been implicated as risk factors for erectile dysfunction (ED), but the results of genetic association studies... (Meta-Analysis)
Meta-Analysis Review
PURPOSE
Endothelial nitric oxide synthase (eNOS) polymorphisms have been implicated as risk factors for erectile dysfunction (ED), but the results of genetic association studies are inconclusive. We performed a meta-analysis of published studies investigating the association between ED and three eNOS polymorphisms, intron 4 VNTR, G894T and T786C in humans.
METHODS
The PubMed, Web of Science, CNKI and Google Scholar databases were searched for relevant studies published up to November 2017. Association studies with case-control design were included. For each study with genotype information we calculated odds ratios (OR) and 95% confidence intervals (CI).
RESULTS
The search identified 13 eligible studies. The G894T and T786C polymorphisms showed a significant association with ED risk in Caucasians (GT + TT versus GG for G894T: OR = 2.13, 95% CI = 1.08-4.19; CC versus CT + TT for T786C: OR = 3.29, 95% CI = 2.30-4.72) and Asians (GT + TT versus GG for G894T: OR = 2.08, 95% CI = 1.53-2.84; CC + CT versus TT for T786C: OR = 3.13, 95% CI = 1.35-7.25). In addition, the intron 4 VNTR polymorphism was associated with ED risk only among Caucasian subjects (aa versus bb + ab: OR = 2.38, 95% CI = 1.15-4.93). We found no evidence of publication bias. The robustness of overall analyses was ensured in sensitivity analyses excluding studies deviating from Hardy-Weinberg equilibrium.
CONCLUSION
Our findings suggest that common genetic polymorphisms in the eNOS gene contribute to risk of ED, presumably by effects on eNOS activity and NO availability.
Topics: Case-Control Studies; Erectile Dysfunction; Genetic Association Studies; Genetic Predisposition to Disease; Genotype; Humans; Male; Minisatellite Repeats; Nitric Oxide Synthase Type III; Odds Ratio; Polymorphism, Genetic; Risk Factors; White People
PubMed: 29654965
DOI: 10.1016/j.ijsu.2018.04.012 -
PLoS Genetics Aug 2020Tribbles homolog 3 (TRIB3) is pseudokinase involved in intracellular regulatory processes and has been implicated in several diseases. In this article, we report that...
Tribbles homolog 3 (TRIB3) is pseudokinase involved in intracellular regulatory processes and has been implicated in several diseases. In this article, we report that human TRIB3 promoter contains a 33-bp variable number tandem repeat (VNTR) and characterize the heterogeneity and function of this genetic element. Analysis of human populations around the world uncovered the existence of alleles ranging from 1 to 5 copies of the repeat, with 2-, 3- and 5-copy alleles being the most common but displaying considerable geographical differences in frequency. The repeated sequence overlaps a C/EBP-ATF transcriptional regulatory element and is highly conserved, but not repeated, in various mammalian species, including great apes. The repeat is however evident in Neanderthal and Denisovan genomes. Reporter plasmid experiments in human cell culture reveal that an increased copy number of the TRIB3 promoter 33-bp repeat results in increased transcriptional activity. In line with this, analysis of whole genome sequencing and RNA-Seq data from human cohorts demonstrates that the copy number of TRIB3 promoter 33-bp repeats is positively correlated with TRIB3 mRNA expression level in many tissues throughout the body. Moreover, the copy number of the TRIB3 33-bp repeat appears to be linked to known TRIB3 eQTL SNPs as well as TRIB3 SNPs reported in genetic association studies. Taken together, the results indicate that the promoter 33-bp VNTR constitutes a causal variant for TRIB3 expression variation between individuals and could underlie the results of SNP-based genetic studies.
Topics: Cell Cycle Proteins; Estonia; Female; Gene Expression Regulation; Genetic Heterogeneity; Genetics, Population; Genotype; Humans; Male; Minisatellite Repeats; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; RNA-Seq; Repressor Proteins; Whole Genome Sequencing
PubMed: 32745133
DOI: 10.1371/journal.pgen.1008981 -
Memorias Do Instituto Oswaldo Cruz 2021Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis... (Review)
Review
Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis transmission in Brazil through genotyping on 56 studies published from 1996-2019 was performed. The clustering rate for mycobacterial interspersed repetitive units - variable tandem repeats (MIRU-VNTR) of 1,613 isolates were: 73%, 33% and 28% based on 12, 15 and 24-loci, respectively; while for RFLP-IS6110 were: 84% among prison population in Rio de Janeiro, 69% among multidrug-resistant isolates in Rio Grande do Sul, and 56.2% in general population in São Paulo. These findings could improve tuberculosis (TB) surveillance and set up a solid basis to build a database of Mycobacterium genomes.
Topics: Bacterial Typing Techniques; Brazil; Genotype; Humans; Minisatellite Repeats; Molecular Epidemiology; Mycobacterium tuberculosis; Polymorphism, Restriction Fragment Length; Whole Genome Sequencing
PubMed: 33729319
DOI: 10.1590/0074-02760200517 -
International Journal of Infectious... Jul 2020Clostridioides difficile is the major cause of infectious nosocomial diarrhoea in industrialized nations. Data on the occurrence of C. difficile in Africa, ribotype (RT)...
BACKGROUND
Clostridioides difficile is the major cause of infectious nosocomial diarrhoea in industrialized nations. Data on the occurrence of C. difficile in Africa, ribotype (RT) distribution, antimicrobial susceptibility patterns and potential zoonotic transmission are scarce.
METHODS
80 Zimbabwean C. difficile isolates from different sources (chicken [n=30], soil [n=21] and humans [n=29]) were investigated using ribotyping, toxin gene detection, resistance testing, multiple-locus variable-number tandem repeat analysis (MLVA), and whole genome sequencing (WGS).
RESULTS
Among chicken isolates, the most common RTs were RT103 (6/30), RT025 (5/30) and RT070 (4/30). Within soil samples, RT025 and RT056 were most common (3/21 each). In contrast, the non-toxigenic RT084 was most frequently found in human isolates (4/29). Toxin genes were detected in only 19/29 human isolates. Susceptibility testing showed no resistance against metronidazole and vancomycin, and resistance against macrolides and rifampicin was scarce (3/80 and 2/80, respectively); however, 26/80 isolates showed moxifloxacin resistance. MLVA and WGS of strains with identical RTs stemming from different sources revealed clustering of RT025 and RT084 isolates from human und non-human samples.
CONCLUSION
No "hypervirulent" strains were found. The detected clusters between human, chicken and soil isolates indicate ongoing transmission between humans and environmental sources and might point towards a zoonotic potential.
Topics: Animals; Chickens; Clostridioides difficile; Drug Resistance, Bacterial; Humans; Minisatellite Repeats; Ribotyping; Soil Microbiology; Zimbabwe
PubMed: 32311450
DOI: 10.1016/j.ijid.2020.04.026 -
Andrologia Nov 2016CAG-repeat in the polymerase γ (POLG) gene encoding polymerase γ for mitochondria is important to spermatogenesis. Compared with a few researchers who raised... (Meta-Analysis)
Meta-Analysis
CAG-repeat in the polymerase γ (POLG) gene encoding polymerase γ for mitochondria is important to spermatogenesis. Compared with a few researchers who raised alteration of CAG-repeat-affected male reproductive ability, others did not find the association between CAG-repeat polymorphisms and male infertility. Therefore, a comprehensive meta-analysis is necessary to determine the association; 13 case-control studies were screened out using keywords search. From these studies, characteristics were extracted for conducting meta-analysis. Odds ratio (OR) and 95% confidence interval (CI) were used to describe the results; the results indicated that CAG-repeat allele was not a risk factor to male infertility (pooled OR = 1.03, 95% CI: 0.79-1.34, P = 0.828). Four different genetic comparisons also demonstrated a negative result: heterozygote comparison (not 10/10 versus 10/10. Pooled OR = 0.99, 95% CI: 0.77-1.27, P = 0.948), homozygote comparison (not 10/not 10 versus 10/10. Pooled OR = 1.08, 95% CI: 0.56-2.06, P = 0.816), the recessive genetic comparison (not 10/not 10 versus not 10/10 + 10/10. Pooled OR = 1.07, 95% CI: 0.58-1.95, P = 0.829) and the dominant genetic comparison (not 10/not 10 + not 10/10 versus 10/10. Pooled OR = 0.97, 95% CI: 0.72-1.29, P = 0.804); based on current researches, this meta-analysis demonstrated no apparent association between POLG-CAG-repeat and male infertility. Similarly, CAG-repeat was not a sensitive site to male infertility.
Topics: Case-Control Studies; DNA Polymerase gamma; DNA-Directed DNA Polymerase; Genetic Association Studies; Humans; Infertility, Male; Male; Minisatellite Repeats; Models, Genetic; Odds Ratio; Polymorphism, Genetic; Trinucleotide Repeats
PubMed: 26790834
DOI: 10.1111/and.12526 -
Veterinary Parasitology Jul 2016A population genetic study of Theileria parva was conducted on 103 cattle and 30 buffalo isolates from Kibaha, Lushoto, Njombe Districts and selected National parks in...
A population genetic study of Theileria parva was conducted on 103 cattle and 30 buffalo isolates from Kibaha, Lushoto, Njombe Districts and selected National parks in Tanzania. Bovine blood samples were collected from these study areas and categorized into 5 populations; Buffalo, Cattle which graze close to buffalo, Kibaha, Lushoto and Njombe. Samples were tested by nested PCR for T. parva DNA and positives were compared for genetic diversity to the T. parva Muguga vaccine reference strain, using 3micro and 11 minisatellite markers selected from all 4 chromosomes of the parasite genome. The diversity across populations was determined by the mean number of different alleles, mean number of effective alleles, mean number of private allele and expected heterozygosity. The mean number of allele unique to populations for Cattle close to buffalo, Muguga, Njombe, Kibaha, Lushoto and Buffalo populations were 0.18, 0.24, 0.63, 0.71, 1.63 and 3.37, respectively. The mean number of different alleles ranged from 6.97 (Buffalo) to 0.07 (Muguga). Mean number of effective alleles ranged from 4.49 (Buffalo) to 0.29 (Muguga). The mean expected heterozygosity were 0.07 0.29, 0.45, 0.48, 0.59 and 0.64 for Muguga, cattle close to buffalo, Kibaha, Njombe, Lushoto and Buffalo populations, respectively. The Buffalo and Lushoto isolates possessed a close degree of diversity in terms of mean number of different alleles, effective alleles, private alleles and expected heterozygosity. The study revealed more diversity in buffalo isolates and further studies are recommended to establish if there is sharing of parasites between cattle and buffaloes which may affect the effectiveness of the control methods currently in use.
Topics: Animals; Buffaloes; Cattle; Genetic Variation; Genetics, Population; Microsatellite Repeats; Minisatellite Repeats; Tanzania; Theileria parva; Theileriasis
PubMed: 27270385
DOI: 10.1016/j.vetpar.2016.04.038 -
BMC Ophthalmology Jan 2019It has been previously reported that one copy of the variable number tandem repeat (VNTR) B alleles of the GPIbα gene increases the risk of non-arteritic ischaemic...
BACKGROUND
It has been previously reported that one copy of the variable number tandem repeat (VNTR) B alleles of the GPIbα gene increases the risk of non-arteritic ischaemic optic neuropathy (NAION) and the second eye involvement. This is the first case where the presence of both alleles is associated with bilateral NAION.
CASE PRESENTATION
A 52-year-old male presented with loss of vision in one eye and was diagnosed with NAION. The following year, he suffered another attack of NAION in the fellow eye. Genetic testing showed that he had both copies of VNTR B alleles of the GPIbα gene.
CONCLUSIONS
We report a case of bilateral NAION in the presence of two copies of VNTR B alleles of the GPIbα gene. This may have further implications for the function of platelet glycoproteins.
Topics: Alleles; Humans; Male; Middle Aged; Minisatellite Repeats; Optic Neuropathy, Ischemic; Platelet Glycoprotein GPIb-IX Complex
PubMed: 30616677
DOI: 10.1186/s12886-018-1010-0 -
Nutrients Jul 2020The dopamine D4 receptor (DRD4) has a predominant expression in the prefrontal cortex (PFC), brain area strictly involved in the modulation of reward processes related... (Review)
Review
The dopamine D4 receptor (DRD4) has a predominant expression in the prefrontal cortex (PFC), brain area strictly involved in the modulation of reward processes related to both food and drug consumption. Additionally, the human DRD4 gene is characterized by a variable number of tandem repeats (VNTR) in the exon 3 and, among the polymorphic variants, the 7-repeat (7R) allele appears as a contributing factor in the neurobiological mechanisms underlying drug abuse, aberrant eating behaviors and related comorbidities. The 7R variant encodes for a receptor with a blunted intracellular response to dopamine, and carriers of this polymorphism might be more tempted to enhance dopamine levels in the brain, through the overconsumption of drugs of abuse or palatable food, considering their reinforcing properties. Moreover, the presence of this polymorphism seems to increase the susceptibility of individuals to engage maladaptive eating patterns in response to negative environmental stimuli. This review is focused on the role of DRD4 and DRD4 genetic polymorphism in these neuropsychiatric disorders in both clinical and preclinical studies. However, further research is needed to better clarify the complex DRD4 role, by using validated preclinical models and novel compounds more selective for DRD4.
Topics: Alleles; Animals; Brain; Dopamine; Exons; Feeding and Eating Disorders; Genetic Predisposition to Disease; Humans; Minisatellite Repeats; Polymorphism, Genetic; Receptors, Dopamine D4; Substance-Related Disorders
PubMed: 32751662
DOI: 10.3390/nu12082288 -
International Journal of... 2021Paratuberculosis is an enteric disease caused by Mycobacterium avium sp. paratuberculosis (MAP) that affects mainly ruminant producing losses to the livestock industry....
Genetic diversity of sp. paratuberculosis by mycobacterial interspersed repetitive Unit-Variable number tandem repeat and multi-locus short-sequence repeat one-sentence summary: Genetic diversity of sp. paratuberculosis isolates.
BACKGROUND
Paratuberculosis is an enteric disease caused by Mycobacterium avium sp. paratuberculosis (MAP) that affects mainly ruminant producing losses to the livestock industry. Many molecular epidemiological methods have been used to discriminate MAP isolates.
METHOD
The aim of this study was to describe the genetic diversity of the Argentinean MAP isolates using a combination of two molecular systems, the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) ("automated and "non-automated") and the multi-locus short-sequence repeat (MLSSR) system.
RESULTS
Thirty-two isolates were identified as MAP of C type by IS900 polymerase chain reaction (PCA) and IS1311 PCA-restriction enzyme analysis. The main patterns found by both MIRU-VNTR systems were INMV1 (54.5%), INMV2 (24.2%) and INMV11 (9.1%). The INMV5, INMV8 and INMV16 were represented with one isolate each (3.0%). Only 4 MIRU-VNTR loci were polymorphic.
CONCLUSION
Those isolates sharing the same INMV patterns were analyzed by MLSSR, being locus 2 the most polymorphic one showing isolates with 9, 10, 11, and more than 11 "G" repeats. Besides, the global discriminatory power among isolates could be increased using both techniques. Based on these results, a short version of the "automated" MIRU-VNTR could be used as a screening tool to group isolates genetically related and subsequently perform the SSR using locus 2 on those isolates sharing the same INMV pattern.
Topics: Animals; Bacterial Typing Techniques; Genetic Variation; Genotype; Humans; Minisatellite Repeats; Mycobacterium avium subsp. paratuberculosis; Paratuberculosis
PubMed: 33707372
DOI: 10.4103/ijmy.ijmy_229_20