Displaying publications 61 - 80 of 106 in total

Abstract:
Sort:
  1. Osada N, Nakagome S, Mano S, Kameoka Y, Takahashi I, Terao K
    Genetics, 2013 Nov;195(3):1027-35.
    PMID: 24026095 DOI: 10.1534/genetics.113.156703
    The ratio of genetic diversity on X chromosomes relative to autosomes in organisms with XX/XY sex chromosomes could provide fundamental insight into the process of genome evolution. Here we report this ratio for 24 cynomolgus monkeys (Macaca fascicularis) originating in Indonesia, Malaysia, and the Philippines. The average X/A diversity ratios in these samples was 0.34 and 0.20 in the Indonesian-Malaysian and Philippine populations, respectively, considerably lower than the null expectation of 0.75. A Philippine population supposed to derive from an ancestral population by founding events showed a significantly lower ratio than the parental population, suggesting a demographic effect for the reduction. Taking sex-specific mutation rate bias and demographic effect into account, expected X/A diversity ratios generated by computer simulations roughly agreed with the observed data in the intergenic regions. In contrast, silent sites in genic regions on X chromosomes showed strong reduction in genetic diversity and the observed X/A diversity ratio in the genic regions cannot be explained by mutation rate bias and demography, indicating that natural selection also reduces the level of polymorphism near genes. Whole-genome analysis of a female cynomolgus monkey also supported the notion of stronger reduction of genetic diversity near genes on the X chromosome.
    Matched MeSH terms: DNA/genetics
  2. Thavanathan J, Huang NM, Thong KL
    Biosens Bioelectron, 2014 May 15;55:91-8.
    PMID: 24368225 DOI: 10.1016/j.bios.2013.11.072
    The unique property of gold nanoparticles (Au NP) to induce colour change and the versatility of graphene oxides (GO) in surface modification makes them ideal in the application of colorimetric biosensor. Thus we developed a label free optical method to detect DNA hybridization through a visually observed colour change. The Au NP is conjugated to a DNA probe and is allowed to hybridize with the DNA target to the GO thus causing a change in colour from pinkish-red to purplish blue. Spectrophometry analysis gave a wavelength shift of 22 nm with 1 µM of DNA target. Sensitivity testing using serially diluted DNA conjugated GO showed that the optimum detection was at 63 nM of DNA target with the limit at 8 nM. This proves the possibility for the detection of DNA hybridization through the use of dual nanoparticle system by visual observation.
    Matched MeSH terms: DNA/genetics*
  3. Abdul Murad NA, Razak ZA, Hussain RM, Syed Hussain SN, Ko Ching Huat C, Che Md Ali SA, et al.
    Asian Pac J Cancer Prev, 2013;14(3):1655-9.
    PMID: 23679251
    BACKGROUND: HER-2/neu is a proto-oncogene that encodes a transmembrane tyrosine kinase growth factor which is crucial for stimulating growth and cellular motility. Overexpression of HER-2/neu is observed in 10-35% of human breast cancers and is associated with pathogenesis, prognosis as well as response to therapy. Given the imperative role of HER-2/neu overexpression in breast cancer, it is important to determine the magnitude of amplification which may facilitate a better prognosis as well as personalized therapy in affected patients. In this study, we determined HER-2/neu protein expression by immunohistochemistry (IHC) concurrently with HER-2/neu DNA amplification by quantitative real time-polymerase chain reaction (Q-PCR).

    MATERIALS AND METHODS: A total of 53 paired tissue samples from breast cancer patients were frozen-sectioned to characterize the tumour and normal tissues. Only tissues with 80% tumour cells were used in this study. For confirmation, Q-PCR was used to determine the HER-2/neu DNA amplification.

    RESULTS: We found 20/53 (37.7%) of the tumour tissues to be positive for HER-2/neu protein overexpression using IHC. Out of these twenty, only 9/53 (17%) cases were in agreement with the Q-PCR results. The concordance rate between IHC and Q-PCR was 79.3%. Approximately 20.7% of positive IHC cases showed no HER-2/neu gene amplification using Q-PCR.

    CONCLUSION: In conclusion, IHC can be used as an initial screening method for detection of the HER-2/neu protein overexpression. Techniques such as Q-PCR should be employed to verify the IHC results for uncertain cases as well as determination of HER-2/neu gene amplification.

    Matched MeSH terms: DNA/genetics
  4. Marimuthu C, Tang TH, Tominaga J, Tan SC, Gopinath SC
    Analyst, 2012 Mar 21;137(6):1307-15.
    PMID: 22314701 DOI: 10.1039/c2an15905h
    The discovery that synthetic short chain nucleic acids are capable of selective binding to biological targets has made them to be widely used as molecular recognition elements. These nucleic acids, called aptamers, are comprised of two types, DNA and RNA aptamers, where the DNA aptamer is preferred over the latter due to its stability, making it widely used in a number of applications. However, the success of the DNA selection process through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments is very much dependent on its most critical step, which is the conversion of the dsDNA to ssDNA. There is a plethora of methods available in generating ssDNA from the corresponding dsDNA. These include asymmetric PCR, biotin-streptavidin separation, lambda exonuclease digestion and size separation on denaturing-urea PAGE. Herein, different methods of ssDNA generation following the PCR amplification step in SELEX are reviewed.
    Matched MeSH terms: DNA/genetics
  5. Ali ME, Hashim U, Mustafa S, Man YB, Yusop MH, Bari MF, et al.
    Nanotechnology, 2011 May 13;22(19):195503.
    PMID: 21430321 DOI: 10.1088/0957-4484/22/19/195503
    We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 °C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 µg ml(-1) swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.
    Matched MeSH terms: DNA/genetics*
  6. Chang YM, Swaran Y, Phoon YK, Sothirasan K, Sim HT, Lim KB, et al.
    Forensic Sci Int Genet, 2009 Jun;3(3):e77-80.
    PMID: 19414156 DOI: 10.1016/j.fsigen.2008.07.007
    17 Y-STRs (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635 or Y-GATA C4, DYS392, Y-GATA H4, DYS437, DYS438 and DYS448) have been analyzed in 320 male individuals from Sarawak, an eastern state of Malaysia on the Borneo island using the AmpFlSTR Y-filer (Applied Biosystems, Foster City, CA). These individuals were from three indigenous ethnic groups in Sarawak comprising of 103 Ibans, 113 Bidayuhs and 104 Melanaus. The observed 17-loci haplotypes and the individual allele frequencies for each locus were estimated, whilst the locus diversity, haplotype diversity and discrimination capacity were calculated in the three groups. Analysis of molecular variance (AMOVA) indicated that 87.6% of the haplotypic variation was found within population and 12.4% between populations (fixation index F(ST)=0.124, p=0.000). This study has revealed that the indigenous populations in Sarawak are distinctly different to each other, and to the three major ethnic groups in Malaysia (Malays, Chinese and Indians), with the Melanaus having a strikingly high degree of shared haplotypes within. There are rare unusual variants and microvariants that were not present in Malaysian Malay, Chinese or Indian groups. In addition, occurrences of DYS385 duplications which were only noticeably present in Chinese group previously was also observed in the Iban group whilst null alleles were detected at several Y-loci (namely DYS19, DYS392, DYS389II and DYS448) in the Iban and Melanau groups.
    Matched MeSH terms: DNA/genetics
  7. Hanafi A, Nograles N, Abdullah S, Shamsudin MN, Rosli R
    J Pharm Sci, 2013 Feb;102(2):617-26.
    PMID: 23192729 DOI: 10.1002/jps.23389
    Cellulose acetate phthalate (CAP) microcapsules were formulated to deliver plasmid DNA (pDNA) to the intestines. The microcapsules were characterized and were found to have an average diameter of 44.33 ± 30.22 μm, and were observed to be spherical with smooth surface. The method to extract pDNA from CAP was modified to study the release profile of the pDNA. The encapsulated pDNA was found to be stable. Exposure to the acidic and basic pH conditions, which simulates the pH environment in the stomach and the intestines, showed that the release occurred in a stable manner in the former, whereas it was robust in the latter. The loading capacity and encapsulation efficiency of the microcapsules were low but the CAP recovery yield was high which indicates that the microcapsules were efficiently formed but the loading of pDNA can be improved. In vitro transfection study in 293FT cells showed that there was a significant percentage of green-fluorescent-protein-positive cells as a result of efficient transfection from CAP-encapsulated pDNA. Biodistribution studies in BALB/c mice indicate that DNA was released at the stomach and intestinal regions. CAP microcapsules loaded with pDNA, as described in this study, may be useful for potential gene delivery to the intestines for prophylactic or therapeutic measures for gastrointestinal diseases.
    Matched MeSH terms: DNA/genetics
  8. Bagheri A, Kamalidehghan B, Haghshenas M, Azadfar P, Akbari L, Sangtarash MH, et al.
    Drug Des Devel Ther, 2015;9:2627-34.
    PMID: 25999696 DOI: 10.2147/DDDT.S79709
    The presence of polymorphisms in the CYP2D6 gene may modulate enzyme level and activity, thereby affecting individual responses to pharmacological treatment. Here, we compared the prevalence of the CYP2D6*10, *4, and 14* alleles in an Iranian population of different ethnicities with those of other populations. Allele and genotype frequency distributions of CYP2D6*10 variants and predicted phenotypes including extensive metabolizers, intermediate metabolizers, and poor metabolizers were analysed in blood samples of 300 unrelated healthy individuals in an Iranian population using polymerase chain reaction (PCR)-restriction fragment length polymorphism, PCR-single-strand conformation polymorphism, and direct genomic DNA sequencing. The CYP2D6*4 (G1846A) and *14 (G1758A) allelic frequencies were not detected in different ethnicities, demonstrating the absence of a significant contribution of these alleles in Iranian populations. However, the T/T, C/T, and C/C genotype frequencies of the CYP2D6*10 allele were significantly different (P<0.01) in all Iranian ethnic groups. Additionally, the frequency of the homozygous T/T variant of the CYP2D6*10 allele was significantly high in the Lure (P<0.017) and low in the Kurd (P<0.002) ethnicities. The frequency of the T/T variant of the CYP2D6*10 allele in central Iran was the highest (P<0.001), while the south of Iran had the lowest frequency (P<0.001). The frequency of the C/T variant of the CYP2D6*10 allele was significantly a bit high (P<0.001) in females compare to males, while the frequencies of the T/T variant in females is similar to males, which are 24.4% and 24.3%, respectively. In contrast to absence of the CYP2D6*4 (G1846A) and *14 (G1758A) alleles in Iranian populations of different ethnicities, the prediction of the CYP2D6*10 allele is required in drug research and routine treatment, where the information would be helpful for clinicians to optimize therapy or identify persons at risk of adverse drug reactions before clinical trials. Approximately 39.3% of subjects (24.3% homozygous T/T CYP2D6*10 as poor metabolizers and 15% heterozygous C/T CYP2D6*10 as intermediate metabolizers) had this allele; therefore, the harmful effects of drugs are relatively common among Iranians.
    Matched MeSH terms: DNA/genetics
  9. Ramanathan S, Gopinath SCB, Arshad MKM, Poopalan P, Anbu P, Lakshmipriya T, et al.
    Sci Rep, 2019 11 19;9(1):17013.
    PMID: 31745155 DOI: 10.1038/s41598-019-53573-9
    Lung cancer is one of the most serious threats to human where 85% of lethal death caused by non-small cell lung cancer (NSCLC) induced by epidermal growth factor receptor (EGFR) mutation. The present research focuses in the development of efficient and effortless EGFR mutant detection strategy through high-performance and sensitive genosensor. The current amplified through 250 µm sized fingers between 100 µm aluminium electrodes indicates the voltammetry signal generated by means of the mutant DNA sequence hybridization. To enhance the DNA immobilization and hybridization, ∼25 nm sized aluminosilicate nanocomposite synthesized from the disposed joss fly ash was deposited on the gaps between aluminium electrodes. The probe, mutant (complementary), and wild (single-base pair mismatch) targets were designed precisely from the genomic sequences denote the detection of EGFR mutation. Fourier-transform Infrared Spectroscopy analysis was performed at every step of surface functionalization evidences the relevant chemical bonding of biomolecules on the genosensor as duplex DNA with peak response at 1150 cm-1 to 1650 cm-1. Genosensor depicts a sensitive EGFR mutation as it is able to detect apparently at 100 aM mutant against 1 µM DNA probe. The insignificant voltammetry signal generated with wild type strand emphasizes the specificity of genosensor in the detection of single base pair mismatch. The inefficiency of genosensor in detecting EGFR mutation in the absence of aluminosilicate nanocomposite implies the insensitivity of genosensing DNA hybridization and accentuates the significance of aluminosilicate. Based on the slope of the calibration curve, the attained sensitivity of aluminosilicate modified genosensor was 3.02E-4 A M-1. The detection limit of genosensor computed based on 3σ calculation, relative to the change of current proportional to the logarithm of mutant concentration is at 100 aM.
    Matched MeSH terms: DNA/genetics
  10. Laosombat V, Fucharoen SP, Panich V, Fucharoen G, Wongchanchailert M, Sriroongrueng W, et al.
    Am J Hematol, 1992 Nov;41(3):194-8.
    PMID: 1415194
    A total of 103 beta thalassemia genes from 78 children (45 with Hb E/beta thalassemia, 8 with beta thalassemia heterozygotes, and 25 with homozygous beta thalassemia) were analyzed using dot-blot hybridization of the polymerase chain reaction-amplified DNA and direct DNA sequencing. Nine mutations were characterized in 98/103 (95%) of beta thalassemia alleles, of which six (a 4 bp deletion in codons 41-42, a G-C transition at position 5 of IVS-1, A-G transition at codon 19, an A-T transition at codon 17, an A-G transition at position -28 upstream of the beta globin gene, a G-T transition at position 1 of IVS-1), accounted for 92%. The spectrum of beta thalassemia mutations in Chinese Thai is similar to that reported among the Chinese from other parts of the world. The distribution of beta thalassemia mutations in Muslim Thai is similar to that reported among Malaysians. The most common beta thalassemia mutation in Thai and Chinese Thai patients is the frameshift mutation at codons 41-42, in comparison with the Muslim Thai in whom the G-C transition at position 5 of the IVS-1 mutation predominates. The heterogeneity of molecular defects causing beta thalassemia should aid in the planning of a prenatal diagnosis program for beta thalassemia in the South of Thailand.
    Matched MeSH terms: DNA/genetics
  11. Iwai K, Hirono A, Matsuoka H, Kawamoto F, Horie T, Lin K, et al.
    Hum Genet, 2001 Jun;108(6):445-9.
    PMID: 11499668
    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a heterogeneous enzyme abnormality with high frequency in tropical areas. We performed population screening and molecular studies of G6PD variants to clarify their distribution and features in Southeast Asia. A total of 4317 participants (2019 males, 2298 females) from 16 ethnic groups in Myanmar, Lao in Laos, and Amboinese in Indonesia were screened with a single-step screening method. The prevalence of G6PD-deficient males ranged from 0% (the Akha) to 10.8% (the Shan). These G6PD-deficient individuals and 12 G6PD-deficient patients who had been diagnosed at hospitals in Indonesia and Malaysia were subjected to molecular analysis by a combination of polymerase-chain-reaction-based single-strand conformation polymorphism analysis and direct sequencing. Ten different missense mutations were identified in 63 G6PD-deficient individuals (50 hemizygotes, 11 heterozygotes, and 2 homozygotes) from 14 ethnic groups. One missense mutation (1291 G-->A) found in an Indonesian Chinese, viz., G6PD Surabaya, was previously unknown. The 487 G-->A (G6PD Mahidol) mutation was widely seen in Myanmar, 383 T-->C (G6PD Vanua Lava) was specifically found among Amboinese, 871 G-->A (G6PD Viangchan) was observed mainly in Lao, and 592 C-->T (G6PD Coimbra) was found in Malaysian aborigines (Orang Asli). The other five mutations, 95 A-->G (G6PD Gaohe), 1003 G-->A (G6PD Chatham), 1360 C-->T (G6PD Union), 1376 G-->T (G6PD Canton), and 1388 G-->A (G6PD Kaiping) were identified mostly in accordance with distributions reported previously.
    Matched MeSH terms: DNA/genetics
  12. Mahil SK, Twelves S, Farkas K, Setta-Kaffetzi N, Burden AD, Gach JE, et al.
    J Invest Dermatol, 2016 11;136(11):2251-2259.
    PMID: 27388993 DOI: 10.1016/j.jid.2016.06.618
    Prominent skin involvement is a defining characteristic of autoinflammatory disorders caused by abnormal IL-1 signaling. However, the pathways and cell types that drive cutaneous autoinflammatory features remain poorly understood. We sought to address this issue by investigating the pathogenesis of pustular psoriasis, a model of autoinflammatory disorders with predominant cutaneous manifestations. We specifically characterized the impact of mutations affecting AP1S3, a disease gene previously identified by our group and validated here in a newly ascertained patient resource. We first showed that AP1S3 expression is distinctively elevated in keratinocytes. Because AP1S3 encodes a protein implicated in autophagosome formation, we next investigated the effects of gene silencing on this pathway. We found that AP1S3 knockout disrupts keratinocyte autophagy, causing abnormal accumulation of p62, an adaptor protein mediating NF-κB activation. We showed that as a consequence, AP1S3-deficient cells up-regulate IL-1 signaling and overexpress IL-36α, a cytokine that is emerging as an important mediator of skin inflammation. These abnormal immune profiles were recapitulated by pharmacological inhibition of autophagy and verified in patient keratinocytes, where they were reversed by IL-36 blockade. These findings show that keratinocytes play a key role in skin autoinflammation and identify autophagy modulation of IL-36 signaling as a therapeutic target.
    Matched MeSH terms: DNA/genetics*
  13. Goh PT, Kuah MK, Chew YS, Teh HY, Shu-Chien AC
    Fish Physiol Biochem, 2020 Aug;46(4):1349-1359.
    PMID: 32239337 DOI: 10.1007/s10695-020-00793-w
    Fish are a major source of beneficial n-3 LC-PUFA in human diet, and there is considerable interest to elucidate the mechanism and regulatory aspects of LC-PUFA biosynthesis in farmed species. Long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis involves the activities of two groups of enzymes, the fatty acyl desaturase (Fads) and elongase of very long-chain fatty acid (Elovl). The promoters of elovl5 elongase, which catalyses the rate-limiting reaction of elongating polyunsaturated fatty acid (PUFA), have been previously described and characterized from several marine and diadromous teleost species. We report here the cloning and characterization of elovl5 promoter from two freshwater fish species, the carnivorous snakehead fish (Channa striata) and zebrafish. Results show the presence of sterol-responsive elements (SRE) in the core regulatory region of both promoters, suggesting the importance of sterol regulatory element-binding protein (Srebp) in the regulation of elovl5 for both species. Mutagenesis luciferase and electrophoretic mobility shift assays further validate the role of SRE for basal transcriptional activation. In addition, several Sp1-binding sites located in close proximity with SRE were present in the snakehead promoter, with one having a potential synergy with SRE in the regulation of elovl5 expression. The core zebrafish elovl5 promoter fragment also directed in vivo expression in the yolk syncytial layer of developing zebrafish embryos.
    Matched MeSH terms: DNA/genetics
  14. Gopinath SCB, Xuan S
    Biotechnol Appl Biochem, 2021 Jun;68(3):554-559.
    PMID: 32460382 DOI: 10.1002/bab.1961
    One of the current issues with thyroid tumor is early diagnosis as it makes the higher possibility of curing. This research was focused to detect and quantify the level of specific target sequence complementation of miR-222 with capture DNA sequence on interdigitated electrode (IDE) sensor. The aluminum electrode with the gap and finger sizes of 10 µm was fabricated on silicon wafer, further the surface was amine-functionalized for accommodating carboxylated-DNA probe. With DNA-target RNA complementation, the detection limit was attained to be 1 fM as estimated by a linear regression analysis [y = 1.5325x - 2.1171 R² = 0.9065] and the sensitivity was at the similar level. Current responses were higher by increasing the target RNA sequence concentrations. Control experiments with mismatched/noncomplementary sequences were failed to complement the capture DNA sequence immobilized on IDE, indicating the specific target validation. This research helps diagnosing and identifying the progression with thyroid tumor and miRNA being a potential "marker" in atypia diagnosis.
    Matched MeSH terms: DNA/genetics*
  15. Caillaud A, de la Iglesia P, Campàs M, Elandaloussi L, Fernández M, Mohammad-Noor N, et al.
    Toxicon, 2010 Feb-Mar;55(2-3):633-7.
    PMID: 19631680 DOI: 10.1016/j.toxicon.2009.07.016
    Protein phosphatase inhibition assay (PPIA), Neuroblastoma cell-based assay (Neuro-2a CBA) and LC-MS/MS analysis revealed for the first time the production of okadaic acid (OA) by a Prorocentrum rhathymum strain. Low amounts of OA were detected by LC-MS/MS analysis. Inhibition of PP2A activity and a weak toxicity to the Neuro-2a CBA were also observed.
    Matched MeSH terms: DNA/genetics
  16. Bahadoran A, Moeini H, Bejo MH, Hussein MZ, Omar AR
    J Pharm Pharm Sci, 2016 Jul-Sep;19(3):325-338.
    PMID: 27806247 DOI: 10.18433/J3G31Q
    PURPOSE: In order to enhance cellular uptake and to facilitate transdermal delivery of DNA vaccine, polyamidoamine (PAMAM) dendrimers conjugated with HIV transactivator of transcription (TAT) was developed.

    METHODS: First, the plasmid DNA (pIRES-H5/GFP) nanoparticle was formulated using PAMAM dendrimer and TAT peptide and then characterized for surface charge, particle size, DNA encapsulation and protection of the pIRES-H5/GFP DNA plasmid to enzymatic digestion. Subsequently, the potency of the TAT-conjugated dendrimer for gene delivery was evaluated through in vitro transfection into Vero cells followed by gene expression analysis including western blotting, fluorescent microscopy and PCR. The effect of the TAT peptide on cellular uptake of DNA vaccine was studied by qRT-PCR and flow cytometry. Finally, the ability of TAT-conjugated PAMAM dendrimer for transdermal delivery of the DNA plasmid was assessed through artificial membranes followed by qRT-PCR and flow cytometry.

    RESULTS: TAT-conjugated PAMAM dendrimer showed the ability to form a compact and nanometre-sized polyplexes with the plasmid DNA, having the size range of 105 to 115 nm and a positive charge of +42 to +45 mV over the N/P ratio of 6:1(+/-).  In vitro transfection analysis into Vero cells confirms the high potency of TAT-conjugated PAMAM dendrimer to enhance the cellular uptake of DNA vaccine.  The permeability value assay through artificial membranes reveals that TAT-conjugated PAMAM has more capacity for transdermal delivery of the DNA compared to unmodified PAMAM dendrimer (P<0.05).

    CONCLUSIONS: The findings of this study suggest that TAT-conjugated PAMAM dendrimer is a promising non-viral vector for transdermal use.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
    Matched MeSH terms: Vaccines, DNA/genetics
  17. Asing, Ali E, Hamid SB, Hossain M, Ahamad MN, Hossain SM, et al.
    PMID: 27643977
    The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected species widely used in exotic foods and traditional medicines. Currently available polymerase chain reaction (PCR) assays to identify MBT lack automation and involve long targets which break down in processed or denatured tissue. This SYBR Green duplex real-time PCR assay has addressed this research gap for the first time through the combination of 120- and 141-bp targets from MBT and eukaryotes for the quantitative detection of MBT DNA in food chain and herbal medicinal preparations. This authentication ensures better security through automation, internal control and short targets that were stable under the processing treatments of foods and medicines. A melting curve clearly demonstrated two peaks at 74.63 ± 0.22 and 78.40 ± 0.31°C for the MBT and eukaryotic products, respectively, under pure, admixed and commercial food matrices. Analysis of 125 reference samples reflected a target recovery of 93.25-153.00%, PCR efficiency of 99-100% and limit of detection of 0.001% under various matrices. The quantification limits were 0.00001, 0.00170 ± 0.00012, 0.00228 ± 0.00029, 0.00198 ± 0.00036 and 0.00191 ± 0.00043 ng DNA for the pure meat, binary mixtures, meatball, burger and frankfurter products, respectively. The assay was used to screen 100 commercial samples of traditional Chinese herbal jelly powder from eight different brands; 22% of them were found to be MBT-positive (5.37 ± 0.50-7.00 ± 0.34% w/w), which was reflected through the Ct values (26.37 ± 0.32-28.90 ± 0.42) and melting curves (74.63-78.65 ± 0.22°C) of the amplified MBT target (120 bp), confirming the speculation that MBT materials are widely used in Chinese herbal desserts, exotic dishes consumed with the hope of prolonging life and youth.
    Matched MeSH terms: DNA/genetics
  18. Asing, Ali ME, Abd Hamid SB, Hossain MA, Mustafa S, Kader MA, et al.
    PLoS One, 2016;11(10):e0163436.
    PMID: 27716792 DOI: 10.1371/journal.pone.0163436
    The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.
    Matched MeSH terms: DNA/genetics
  19. Lai JY, Loh Q, Choong YS, Lim TS
    Biotechniques, 2018 11;65(5):269-274.
    PMID: 30394125 DOI: 10.2144/btn-2018-0031
    Gene assembly methods are an integral part of molecular cloning experiments. The majority of existing vector assembly methods stipulate a need for exonucleases, endonucleases and/or the use of single-stranded DNA as starting materials. Here, we introduced a vector assembly method that employs conventional PCR to amplify stable double-stranded DNA fragments and assembles them into functional vectors specifically for antibody chain shuffling. We successfully formed vectors using cassettes amplified from different templates and assembled an array of single chain fragment variable clones of fixed variable heavy chain, with different variable light chains - a chain shuffling process for antibody maturation. The method provides an easy alternative to the conventional cloning process.
    Matched MeSH terms: DNA/genetics*
  20. Khairil Mokhtar NF, El Sheikha AF, Azmi NI, Mustafa S
    J Sci Food Agric, 2020 Mar 15;100(4):1687-1693.
    PMID: 31803942 DOI: 10.1002/jsfa.10183
    BACKGROUND: The growth of halal food consumption worldwide has resulted in an increase in the request for halal authentication. DNA-based detection using powerful real-time polymerase chain reaction (PCR) technique has been shown to be highly specific and sensitive authentication tool. The efficient DNA extraction method in terms of quality and quantity is a backbone step to obtain successful real-time PCR assays. In this study, different DNA extraction methods using three lysis buffers were evaluated and developed to recommend a much more efficient method as well as achieve a successful detection using real-time PCR.

    RESULTS: The lysis buffer 2 (LB2) has been shown to be the best lysis buffer for DNA extraction from both raw and processed meat samples comparing to other lysis buffers tested. Hence, the LB2 has been found to be ideal to detect meat and porcine DNAs by real-time PCR using pairs of porcine specific primers and universal primers which amplified at 119 bp fragment and 93 bp fragment, respectively. This assay allows detection as low as 0.0001 ng of DNA. Higher efficiency and sensitivity of real-time PCR via a simplified DNA extraction method using LB2 have been observed, as well as a reproducible and high correlation coefficient (R2  = 0.9979) based on the regression analysis of the standard curve have been obtained.

    CONCLUSION: This study has established a fast, simple, inexpensive and efficient DNA extraction method that is feasible for raw and processed meat products. This extraction technique allows an accurate DNA detection by real-time PCR and can also be implemented to assist the halal authentication of various meat-based products available in the market. © 2019 Society of Chemical Industry.

    Matched MeSH terms: DNA/genetics*
Filters
Contact Us

Please provide feedback to Administrator (afdal@afpm.org.my)

External Links