• Title/Summary/Keyword: primer selection

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Development of Nested PCR Primer Set for the Specific and Highly Sensitive Detection of Human Parvovirus B19

  • Cho, Kyu-Bong
    • Biomedical Science Letters
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    • v.24 no.4
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    • pp.390-397
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    • 2018
  • For the specific detection of human Parvovirus B19 (HuPaV-B19), we designed ten specific PCR primers from 3,800~4,500 nucleotides of HuPaV-B19 complete genome (NC_000883.2). Seventeen candidate PCR primer sets for specific detecting HuPaV-B19 were constructed. In specific reaction of HuPaV-B19, seventeen PCR primer sets showed specific band, however five PCR primer sets were selected basis of band intensity, amplicon size and location. In non-specific reaction with seven reference viruses, four PCR primer sets showed non-specific band, however one PCR primer set is not. Detection sensitivity of final selective PCR primer set was $100fg/{\mu}L$ for 112 minute, and PCR amplicon is 539 base pairs (bp). In addition, nested PCR primer set was developed, for detection HuPaV-B19 from a low concentration of contaminated samples. Selection of nested PCR primer set was basis of sensitivity and groundwater sample tests. Detection sensitivity of final selective PCR and nested PCR primer sets for the detection of HuPaV-B19 were $100fg/{\mu}L$ and $100ag/{\mu}L$ basis of HuPaV-B19 plasmid, it was able to rapid and highly sensitive detection of HuPaV-B19 than previous reports. We expect developed PCR primer set in this study will used for detection of HuPaV-B19 in various samples.

RAPD marker를 이용한 참돔 집단의 유전적 특성 분석

  • 장요순;노충환;홍경표;명정구;김종만
    • Proceedings of the Korean Aquaculture Society Conference
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    • 2003.10a
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    • pp.34-34
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    • 2003
  • 한국산 선발계통 및 일본산 양식계통과 이들 두 계통간 잡종 참돔 집단의 유전적 특성을 분석하기 위하여, RAPD (Random Amplified Polymorphic DNA) marker를 탐색하였다. 10개의 염기로 이루어진 200개의 random primer 분석을 통하여 polymorphic pattern을 나타내는 23개의 random primer를 선발하였으며, 각 primer의 재현성을 확인하였다. 이들 중 OPA-11 primer는 크기가 각각 600 bp, 650 bp 및 750 bp 인 3개의 DNA 단편에 의하여 4개의 genotype을 나타냈으며, 각 genotype의 빈도는 집단간차이를 보였고, 한국산 선발계통 집단에서는 4개의 genotype이 모두 발견되는 반면, 일본산 양식계통 및 일본산 양식계통을 포함한 교배집단에서는 특정 genotype만 발견되었다. OPA-11 primer 유래의 polymorphic DNA 단편을 cloning하고 염기서열을 결정하였으며, SCAR (Sequence Characterized Amplified Region) primer를 제작하고 분석하였다. 본 연구는 참돔집단의 유전적 특성 파악 및 집단 구별에 RAPD marker를 활용하였으며, 참돔 육종시 형질 및 기능관련 DNA marker 탐색에 적용하기 위하여, 이후의 연구에서는 SCAR과 RFLP 분석에 RAPD marker를 이용하여 100% 정확도를 갖는 RFLP maker를 찾고, MAS (Marker-Assisted Selection)에 적용하고자 한다.

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A New Multiplex-PCR for Urinary Tract Pathogen Detection Using Primer Design Based on an Evolutionary Computation Method

  • Garcia, Liliana Torcoroma;Cristancho, Laura Maritza;Vera, Erika Patricia;Begambre, Oscar
    • Journal of Microbiology and Biotechnology
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    • v.25 no.10
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    • pp.1714-1727
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    • 2015
  • This work describes a new strategy for optimal design of Multiplex-PCR primer sequences. The process is based on the Particle Swarm Optimization-Simplex algorithm (Mult-PSOS). Diverging from previous solutions centered on heuristic tools, the Mult-PSOS is selfconfigured because it does not require the definition of the algorithm's initial search parameters. The successful performance of this method was validated in vitro using Multiplex-PCR assays. For this validation, seven gene sequences of the most prevalent bacteria implicated in urinary tract infections were taken as DNA targets. The in vitro tests confirmed the good performance of the Mult-PSOS, with respect to infectious disease diagnosis, in the rapid and efficient selection of the optimal oligonucleotide sequences for Multiplex-PCRs. The predicted sequences allowed the adequate amplification of all amplicons in a single step (with the correct amount of DNA template and primers), reducing significantly the need for trial and error experiments. In addition, owing to its independence from the initial selection of the heuristic constants, the Mult-PSOS can be employed by non-expert users in computational techniques or in primer design problems.

Pickprimer: A Graphic User Interface Program for Primer Design on the Gene Target Region (픽프라이머 : 유전자 목표 구간 탐색 모듈을 포함한 프라이머 제작 그래픽 프로그램)

  • Chung, Hee;Mun, Jeong-Hwan;Lee, Seung-Chan;Yu, Hee-Ju
    • Horticultural Science & Technology
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    • v.29 no.5
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    • pp.461-466
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    • 2011
  • In genetic and molecular breeding studies of plants, researchers need to design various kinds of primers based on their research purposes. So far many kinds of web- or script-based non-commercial programs for primer design are available. Because most of them do not include user interface for multipurpose usage including gene structure prediction and direct target selection on sequences, it has been a laborious work to design primers targeting on the exon or intron regions of interesting genes. Here we report a primer designing graphic user interface program, Pickprimer, that includes gene structure prediction and primer design modules by combining source codes of the Spidey and Primer3 programs. This program provides simple graphic user interface to input sequences and design primers. Genomic sequence and mRNA or coding sequence of genes can be copy and pasted or input as fasta or text files. Based on alignment of the input sequences using the Spidey module, a putative gene structure is graphically visualized along with exon-intron sequences of color codes. Primer design can be easily performed by dragging mouse on the displayed sequences or input primer targeting position with desirable values of primers. The output of designed primers with detailed information is provided by the Primer3 module. PCR evaluation of 24 selected primer sets successfully amplified single amplicons from six Brassica rapa cultivars. The Pickprimer will be a convenient tool for genetic and molecular breeding studies of plants.

SSR-Primer Generator: A Tool for Finding Simple Sequence Repeats and Designing SSR-Primers

  • Hong, Chang-Pyo;Choi, Su-Ryun;Lim, Yong-Pyo
    • Genomics & Informatics
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    • v.9 no.4
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    • pp.189-193
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    • 2011
  • Simple sequence repeats (SSRs) are ubiquitous short tandem duplications found within eukaryotic genomes. Their length variability and abundance throughout the genome has led them to be widely used as molecular markers for crop-breeding programs, facilitating the use of marker-assisted selection as well as estimation of genetic population structure. Here, we report a software application, "SSR-Primer Generator " for SSR discovery, SSR-primer design, and homology-based search of in silico amplicons from a DNA sequence dataset. On submission of multiple FASTA-format DNA sequences, those analyses are batch processed in a Java runtime environment (JRE) platform, in a pipeline, and the resulting data are visualized in HTML tabular format. This application will be a useful tool for reducing the time and costs associated with the development and application of SSR markers.

A Study on the Physical Properties Change of Waterproofing Membrane Coating by Application of Modified Polymer Primer (개질 폴리머계 프라이머 적용에 따른 도막 방수재의 물리적 특성 변화 연구)

  • Lee, Jung-Hun;Cho, Hong-Bum;Jeon, Hyun-Soo;Park, Ki-Hong;Kim, Jin-Sik;Oh, Sang-Keun
    • Proceedings of the Korean Institute of Building Construction Conference
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    • 2022.11a
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    • pp.191-192
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    • 2022
  • It was intended to evaluate whether the primer affects the physical performance of the waterproofing membrane coating. For this end, the physical performance change of the urethane waterproofing membrane coating was evaluated for urethane-based and polymer-based primers. As a result of the evaluation, it was confirmed that the type of primer may affect the physical performance of the waterproofing membrane coating. In particular, a difference in performance of more than 100% was confirmed in tensile strength and percentage elongation. Through these results, it was confirmed that the selection of the primer was important. In the future, follow-up studies on various waterproofing membrane coatings are needed.

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Selection of RAPD marker to discriminate the bolting-resistant varieties and commercial dried medicinal materials of Angelica species (당귀 내추대성 품종 및 건재약재 판별을 위한 RAPD marker 선발)

  • Bang, Kyong-Hwan;Yu, Hong-Seob;Koo, Dal-Hoe;Cho, Joon-Hyeong;Park, Hee-Woon;Seong, Nak-Sul;Park, Sang-Il;Kim, Hong-Sig
    • Korean Journal of Medicinal Crop Science
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    • v.10 no.1
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    • pp.46-50
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    • 2002
  • In DNA level, genetic study of Angelica species was firstly conducted to discriminate the bolting-resistant or low bolting variety, so called as Manchu, from other Korea collected lines and also this technuque was applied to identify the origin of commercial dried materials obtained from current oriental medicinal market. By RAPD analysis with 72 primers including sixty of 10-mers and twelve of 20-mers, respectively, three primers, which were related to the bolting resistant traits of Angelica gigas, were identified. Comparing the RAPD bands, URP04 primer showed the 1.7 kb specific band, which seemed to be related to delaying bolting traits, since it was observed only in Jinbu elite lines but not in others. On the other hand, since 1.2 kb band amplified by OPD11 was observed in other collected lines but not in Manchu var. and Jinbu line, this primer also could be considered as a selection marker for identifying bolting resistant or delaying bolting traits. In the same manner, since OPP09 did not show 1 kb major band but produced 0.8 kb and 1.2 kb bands in Manchu var., these three bands amplified by the primer could be considered one of the important key specifying Manchu var. related with the trait of Angelica gigas. OPC02 primer showed the same band patterns in all Korean collected lines, but not in other foreign introduced lines, such as A. sinensis from China, and A. acutiloba from Japan. Since these four RAPD primers, OPD11, OPP09, URP04, and OPC02 showed the specific polymorphisms in Angelica species, thus, these were useful to discriminate the three Angelica species, A. gigas, A. sinensis, and A. acutiloba.

Selection and Genetic Relationship of Salt Tolerant Rice Mutants by in vitro Mutagenesis

  • Song, Jae Young;Kim, Dong Sub;Lee, Myung-Chul;Lee, Kyung Jun;Kim, Jin-Baek;Kim, Sang Hoon;Yun, Song Joong;Kang, Si-Yong
    • Journal of Radiation Industry
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    • v.4 no.4
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    • pp.307-312
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    • 2010
  • Plants have evolved physiological, biochemical and metabolic mechanisms to increase their survival under the adverse conditions. This present study has been performed to select salt-tolerant rice mutant lines through in vivo and in vitro mutagenesis with gamma-rays. For the selection of the salt-tolerant rice mutants, we conducted three times of selection procedure using 1,500 gamma ray mutant lines resulted from an embryo culture of the original rice cv. Dongan (wild-type, WT): first, selection in the a nutrient solution with 171 mM NaCl; second, selection under in vitro condition with 171 mM NaCl; and third, selection in a reclaimed saline land. Based on a growth comparison of the entries, out of the mutant lines, two putative 2 salt tolerant (ST) rice mutant lines, ST-87 and ST-301, were finally selected. The survival rate of the WT, ST-87 and ST-301 were 36.6%, 60% and 66.3% after 7 days in 171 mM NaCl treatment, respectively. The WT and two salt tolerant mutant lines were used to analyze their genetic variations. A total of 21 EcoRI and Msel primer combinations were used to analyze the genetic relationship of among the two salt-tolerant lines and the WT using the ABI3130 capillary electrophoresis system. In the AFLP analysis, a total of 1469 bands were produced by the 21 primer combinations, and 700 (47.6%) of them were identified as having polymorphism. The genetic similarity coefficients were ranged from 0.52 between the ST-87 and WT to 0.24 between the ST-301 and the WT. These rice mutant lines will be used as a control plot for physiological analysis and genetic research on salt tolerance.