• Title/Summary/Keyword: Tandem repeat

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Analysis of Contamination Sources of Staphylococcus aureus Related to Perilla Leaves Using Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA) (Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA)를 이용한 들깻잎 중 Staphylococcus aureus의 오염원분석)

  • Kim, Se-Ri;Shim, Won-Bo;Han, Noo Ri;Chung, Duck-Hwa
    • Journal of Food Hygiene and Safety
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    • v.29 no.4
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    • pp.278-284
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    • 2014
  • To investigate the prevalence of Staphylococcus aureus (S. aureus) during production of perilla leaves, a total of 261 samples, including water, soil, surroundings of cultivation and packing, workers, and perilla leaves, was examined.. To trace the contamination sources of S. aureus related to perilla leaves, MLVA (Multi-Locous Variable number of tandem repeat Analysis), which is a very efficient method to discriminate strains with minimum molecular biology equipment was applied to S. aureus isolated from perilla leaves farms. S. aureus was isolated in perilla leaves from 9 of 38 farms at 0-2.92 log CFU/g. S. aureus was also found in working environment, including packing vinyl, worker clothes, irrigation water and hands. The patterns of MLVA of isolates from perilla leaves matched with those of isolates from packing table, irrigation water, packing vinyl, and hands. The isolates were successfully examined and determined by MLVA, thus elucidating S. aureus source and spread.

Detection of Fragment Length Polymorphism of the VNTR Loci D1S80 and D2S123 by PCR Amplification, PAGE and Silver Staining

  • Nam, Hyun-Suk;Kim, Eun-Hee;Yoon, Wan-Hee;Lee, Kong-Joo
    • BMB Reports
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    • v.28 no.4
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    • pp.359-362
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    • 1995
  • The highly polymorphic variable number of tandem repeat (VNTR) loci in the human genome are informative markers for the genetic characterization of individuals in the paternity test and forensic science as well as for the study of human disease. In this study, VNTR loci D1S80 and D2S123 have been amplified by PCR and the amplified length polymorphic alleles were detected with a discontinuous vertical PAGE system and silver staining. For explicit DNA typing, PCR optimization, in which amplification efficiencies are similar over a wide range of allele sizes, non-specific amplifications are minimal, and new longer alleles have high amplification efficiency, has been performed by changing the PCR reaction buffer composition and thermal cycling conditions. It turned out that adding an appropriate amount of Tween 20 and NP40 to the PCR reaction buffer and raising the annealing temperature to $68^{\circ}C$ in thermal cycling made it possible for optimal VNTR loci amplification. A modified PAGE system for VNTR separation was established. Under these conditions, new longer alleles in the 01580 locus were discovered and 025123 pattern changes in colorectal tumors were observed. These technical tips are valuable for detecting various amplified fragment length polymorphisms.

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Forensic STR Analysis of Mixed Chimerism after Allogeneic Bone Marrow Transplantation

  • Eom, Yong-Bin
    • Biomedical Science Letters
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    • v.16 no.3
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    • pp.193-196
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    • 2010
  • Multiplex PCR-based short tandem repeat (STR) analysis is considered as a good tool for monitoring bone marrow engraftment after sex-mismatched allogeneic transplantation and provides a sensitive and accurate assessment of the contribution of both donor and/or recipient cells in post-transplantation specimens. Forensic STR analysis and quantitative real time PCR are used to determine the proportion of donor versus recipient each contained within the total DNA. The STR markers were co-amplified in a single reaction by using commercial $PowerPlex^{(R)}$ 16 system and $AmpFISTR^{(R)}$ $Identifiler^{(R)}$ / $Yfiler^{(R)}$ PCR amplification kits. Separation of the PCR products and fluorescence detection were performed by ABI $PRIS^{(R)}$ 3100 Genetic Analyzer with capillary electrophoresis. The $GeneMapper^{TM}$ ID software were used for size calling and analysis of STR profiles. Extracted DNA was quantified by the $Quantifiler^{TM}$ Human DNA / Y Human Male DNA Quantification Kit The intent of this study was to analyze the ratio of donor versus recipient cells in the post-transplant peripheral blood, spleen, lung and kidney specimens. Specimens were taken from the traffic accident male victim who had been engrafted from bone marrow female donor. Blood and spleen specimens displayed female donor DNA profile. Kidney specimen showed male recipient DNA profile. Interestingly, lung tissue showed mixed profiles. The findings of this study indicate that the forensic STR analysis using fluorescence labeling PCR combined with capillary electrophoresis is quick and reliable enough to assess the ratio of donor versus recipient cells and to monitor the mixed chimeric patterns.

Evaluation of DNA Extraction Methods from Low Copy Number (LCN) DNA Samples for Forensic DNA Typing

  • Eom, Yong-Bin
    • Biomedical Science Letters
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    • v.15 no.3
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    • pp.229-232
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    • 2009
  • DNA isolation for PCR-based short tandem repeat (STR) analysis is essential to recover high yields of amplifiable DNA from low copy number (LCN) DNA samples. There are different methods developed for DNA extraction from the small bloodstain and gloves, commonly found at crime scenes. In order to obtain STR profiles from LCN DNA samples, DNA extraction protocols, namely the automated $iPrep^{TM}$ $ChargeSwitch^{(R)}$ method, the automated $QIAcube^{TM}$ method, the automated $Maxwell^{(R)}$ 16 DNA $IQ^{TM}$ Resin method, and the manual $QIAamp^{(R)}$ DNA Micro Kit method, were evaluated. Extracted DNA was quantified by the $Quantifiler^{TM}$ Human DNA Quantification Kit and DNA profiled by $AmpFISTR^{(R)}$ $Identifiler^{(R)}$ Kit. Results were compared based on the amount of DNA obtained and the completeness of the STR profiles produced. The automated $iPrep^{TM}$ $ChargeSwitch^{(R)}$ and $QIAcube^{TM}$ methoas produced reproducible DNA of sufficient quantity and quality trom the dried blood spot. This two automated methods showed a quantity and quality comparable to those of the forensic manual standard protocols normally used in our laboratory. In our hands, the automated DNA extraction method is another obvious choice when the forensic case sample available is bloodstain. The findings of this study indicate that the manual simple modified $QIAamp^{(R)}$ DNA Micro Kit method is best method to recover high yields of amplifiable DNA from the numerous potential sources of LCN DNA samples.

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Validation of Reduced-volume Reaction in the PowerQuant® System for human DNA Quantification

  • Kim, Hyojeong;Cho, Yoonjung;Kim, Jeongyong;Lee, Ja Hyun;Kim, Hyo Sook;Kim, Eungsoo
    • Biomedical Science Letters
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    • v.26 no.4
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    • pp.275-287
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    • 2020
  • Since its introduction in the forensic field, quantitative PCR (qPCR) has played an essential role in DNA analysis. Quality of DNA should be evaluated before short tandem repeat (STR) profiling to obtain reliable results and reduce unnecessary costs. To this end, various human DNA quantification kits have been developed. Among these kits, the PowerQunat® System was designed not only to determine the total amount of human DNA and human male DNA from a forensic evidence item, but also to offer data about degradation of DNA samples. However, a crucial limitation of the PowerQunat® System is its high cost. Therefore, to minimize the cost of DNA quantification, we evaluated kit performance using a reduced volume of reagents (1/2-volume) using DNA samples of varying types and concentrations. Our results demonstrated that the low-volume method has almost comparable performance to the manufacturer's method for human DNA quantification, human male DNA quantification, and DNA degradation index. Furthermore, using a reduced volume of regents, it is possible to run 2 times more reactions per kit. We expect the proposed low-volume method to cut costs in half for laboratories dealing with large numbers of DNA samples.

A Review of Extended STR Loci and DNA Database

  • Cho, Yoonjung;Lee, Min Ho;Kim, Su Jin;Park, Ji Hwan;Jung, Ju Yeon
    • Biomedical Science Letters
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    • v.28 no.3
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    • pp.157-169
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    • 2022
  • DNA typing is the typical technology in the forensic science and plays a significant role in the personal identification of victims and suspects. Short tandem repeat (STR) is the short tandemly repeated DNA sequence consisting of 2~7 bp DNA units in specific loci. It is disseminated across the human genome and represents polymorphism among individuals. Because polymorphism is a key feature of the application of DNA typing STR analysis, STR analysis becomes the standard technology in forensics. Therefore, the DNA database (DNA-DB) was first introduced with 4 essential STR markers for the application of forensic science; however, the number of STR markers was expanded from 4 to 13 and 13 to 20 later to counteract the continuously increased DNA profile and other needed situations. After applying expanded STR markers to the South Korean DNA-DB system, it positively affected to low copy number analysis that had a high possibility of partial DNA profiles, and especially contributed to the theft cases due to the high portion of touch DNA evidence in the theft case. Furthermore, STR marker expansion not only contributed to the resolution of cold cases but also increased kinship index indicating the potential for improved kinship test accuracy using extended STR markers. Collectively, the expansion of the STR locus was considered to be necessary to keep pace with the continuously increasing DNA profile, and to improve the data integrity of the DNA-DB.