• Title/Summary/Keyword: PCR-amplify

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A Simple and Rapid Gene Amplification from Arabidopsis Leaves Using AnyDirect System

  • Yang, Young-Geun;Kim, Jong-Yeol;Soh, Moon-Soo;Kim, Doo-Sik
    • BMB Reports
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    • v.40 no.3
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    • pp.444-447
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    • 2007
  • Polymerase chain reaction (PCR) is a powerful technique in molecular biology and is widely used in various fields. By amplifying DNA fragments, PCR has facilitated gene cloning procedures, as well as molecular genotyping. However, the extraction of DNA from samples often acts as a limiting step of these reactions. In particular, the extraction of PCR-compatible genomic DNA from higher plants requires complicated processes and tedious work because plant cells have rigid cell walls and contain various endogenous PCR inhibitors, including polyphenolic compounds. We recently developed a novel solution, referred to as AnyDirect, which can amplify target DNA fragments directly from whole blood without the need for DNA extraction. Here, we developed a simple lysis system that could produce an appropriate template for direct PCR with AnyDirect PCR buffer, making possible the direct amplification of DNA fragments from plant leaves. Thus, our experimental procedure provides a simple, convenient, non-hazardous, inexpensive, and rapid process for the amplification of DNA from plant tissue.

A Novel PCR Primers HPU185 and HPL826 Based on 16S rRNA Gene for Detection of Helicobacter pylori

  • Kim, Jong-Bae;Kim, Geun-Hee;Kim, Hong;Jin, Hyun-Seok;Kim, Young-Sam;Ha, Soo-Hyun;Lee, Dong-Ki
    • The Journal of the Korean Society for Microbiology
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    • v.35 no.4
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    • pp.283-288
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    • 2000
  • The PCR primer set JW21-JW22 of Weiss et al. (19), which was reported to amplify a 139-bp fragment of the l6S rRNA gene of Helicobacter pylori, has been recently used for the detection of H. pylori in clinical specimens. However, when we applied JW21-JW22 PCR to other members of the genus Helicobacter and unrelated microorganisms, all of these bacteria produced a 139-bp PCR product. Therefore, we designed a novel primer set, HPU185-HPL826, which produced a 642-bp amplicon of the l6S rRNA gene of H. pylori. Then we further examined the specificity of the novel PCR assay using Southern blot hybridization with an internal probe, HPP225. The PCR assay described in this study was shown to be highly sensitive and specific only to the H. pylori 16S rRNA gene sequences.

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Multiplex PCR Detection for 3 Events of Genetically Modified Maize, DAS-59122-7, TC6275, and MIR604

  • Ahn, Ji-Hye;Kim, Jae-Hwan;Kim, Su-Youn;Lee, Woo-Young;Park, Sun-Hee;Kim, Hae-Yeong
    • Food Science and Biotechnology
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    • v.17 no.3
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    • pp.569-572
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    • 2008
  • A multiplex polymerase chain reaction (PCR) method was developed to simultaneously detect 3 events of genetically modified (GM) maize. The event-specific primers were used to discriminate the following 3 events of GM maize (DAS-59122-7, TC6275, and MIR604) using multiplex PCR method. The zein gene was used as an endogenous maize reference gene in the multiplex PCR detection. The primer pair Zein-FIR producing a 99 bp amplicon was used to amplify the zein gene. The primer JI-Das-F1/R1 for DAS-59122-7, JI-TC6275-F3/R3 for TC6275, and JI-MIR F1/R1 for MIR604 yielded an amplicon of 130, 162, and 197 bp, respectively. The detection limit of multiplex PCR was 1% for DAS-59122-7, TC6275, and MIR604 for one reaction.

Optimization of DNA Extraction from a Single Living Ciliate for Stable and Repetitive PCR Amplification

  • Kim, Se-Joo;Min, Gi-Sik
    • Animal cells and systems
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    • v.13 no.3
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    • pp.351-356
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    • 2009
  • Ciliates are undoubtedly one of the most diverse protozoans that play a significant role in ecology. However, molecular examination, based on comparing the DNA sequences, has been done on a limited number of the species. Because most ciliates are uncultivable and their population sizes are often too small, it is usually difficult to obtain sufficient genomic DNA required for PCR based experiments. In the present study, we evaluated the effectiveness of four commercial DNA extraction procedures that extract high quality genomic DNA from a single ciliate cell. It was discovered that RED Extract-N-$Amp^{TM}$ PCR kit is the best method for removing PCR-inhibiting substances and minimizing DNA loss during purification. This method can also amplify more than 25 reactions of PCR. In addition, this technique was applied to single cells of 19 species belonged to 7 orders under 5 classes that isolated from mixed natural populations. Their small subunit ribosomal DNA (SSU rDNA) was successfully amplified. In summary, we developed a simple technique for the high-yield extraction of purified DNA from a single ciliate cell that may be more useful for rare ciliates, such as tiny and uncultivable marine microbes.

Development and Practical Use of RT-PCR for Seed-transmitted Prune dwarf virus in Quarantine

  • Lee, Siwon;Shin, Yong-Gil
    • The Plant Pathology Journal
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    • v.30 no.2
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    • pp.178-182
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    • 2014
  • Among imported plants, seeds are the items that have many latent pathogens and are difficult to inspect. Also, they are the import and export items whose market is expected to expand. The biggest problem with seeds is viruses. Prune dwarf virus (PDV) is the virus that is commonly inspected in Prunus cerasifera, P. persica, P. armeniaca, P. mandshurica, P. cerasus, P. avium or P. serotina seeds. In this study, two RT-PCR primer sets, which can promptly and specifically diagnose plant quarantine seed-transmitted PDV, were developed; and nested PCR primers, where products amplify 739 and 673 nucleotides (nt), and an nested PCR-product, 305 nt, can be obtained as these products are amplified again, were developed. Also, a modified-positive control plasmid was developed, where the restriction enzyme XhoI, which can identify the contamination of samples from the control, was inserted. The method developed in this study has detected PDV in 18 cases since 2007, and is expected to continuously contribute to the plant quarantine in Korea.

Isolation and PCR detection of Listeria monocytogenes on raw beef and pork carcass (소와 돼지도체에서 Listeria monocytogenes의 분리 및 PCR 검출 방법에 관한 연구)

  • Chae, Hee-Sun;Kim, Doo-Hwan;Kim, Gu-Hyun;Shin, Bang-Woo;Jo, Mi-Yoeng;Kweon, Taek-Boo;Lee, Jung-Hak
    • Korean Journal of Veterinary Service
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    • v.26 no.2
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    • pp.105-111
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    • 2003
  • From February 2000 to December 2001, A total of 1,785 samples was taken from beef and pork carcasses in Seoul. Seven(0.69%) Listeria spp. were isolated from the 1,014 of beef carcasses, and five(0.65%) were isolated from the 771 of pork carcasses. The isolates were identified L monocytogenes by API listeria, and VIDAS LMO kit, serological test and PCR assay were preformed. A total 12 strains of L monocytogenes were isolated form samples tested and all of the strains were classified into serotype 1. PCR primers are selected to amplify a 520-base pair(bp) DNA fragment from the listeolysin O gene(hlyA) of Listeria monocytogenes. A 520-bp product was detected in PCR with DNA from L monocytogenes, but not from the other Listeria species tested.

Development of Reverse Transcription Semi-nested PCR Primer Pairs for the Specific and Highly Sensitive Detection of Human Aichivirus A1

  • Lee, Siwon;Cho, Kyu Bong
    • Biomedical Science Letters
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    • v.25 no.4
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    • pp.331-338
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    • 2019
  • Human Aichivirus A1 (HuAiV-A1) is a waterborne human pathogenic virus classified as Picornaviridae and Kobuvirus. In this study, we developed a method that can detect about 35 minutes faster with the same detection sensitivity level than the previously reported HuAiV-A1 diagnostic RT-PCR primer. The RT-PCR primer sets developed in this study are capable of detecting HuAiV-A1 at a level of about 100 ag and formed 563 bp amplification product. In addition, the RT-nested PCR method was able to amplify 410 bp using the RT-PCR product as a template. The detection sensitivity of our method was 10 times higher than the method with the highest detection sensitivity to date. Therefore, the detection method of HuAiV-A1 developed in this study is expected to be used in the water environment in which a small amount of virus exists. Also, this detection method is expected to be used as HuAiV-A1 diagnostic technology in both clinical and non-clinical field.

Multiplex PCR Detection of 4 Events of Genetically Modified Soybeans (RRS, A2704-12, DP356043-5, and MON89788)

  • Kim, Jae-Hwan;Seo, Young-Ju;Sun, Seol-Hee;Kim, Hae-Yeong
    • Food Science and Biotechnology
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    • v.18 no.3
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    • pp.694-699
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    • 2009
  • A multiplex polymerase chain reaction (PCR) method was developed for the detection of 4 events of genetically modified (GM) soybean. The event-specific primers were designed from 4 events of GM soybean (RRS, A2704-12, DP356043-5, and MON89788). The lectin was used as an endogenous reference gene of soybean in the PCR detection. The primer pair YjLec-4-F/R producing 100 bp amplicon was used to amplify the lectin gene and no amplified product was observed in any of the 9 different plants used as templates. This multiplex PCR method allowed for the detection of event-specific targets in a genomic DNA mixture of up to 1% GM soybean mixture containing RRS, A2704-12, DP356043-5, and MON89788. In this study, 20 soybean products obtained from commercial food markets were analyzed by the multiplex PCR. As a result, 6 samples contained RRS. These results indicate that this multiplex PCR method could be a useful tool for monitoring GM soybean.

Multiplex PCR Detection of the MON1445, MON15985, MON88913, and LLcotton25 Varieties of GM Cotton

  • Kim, Jae-Hwan;Kim, Sun-A;Seo, Young-Ju;Lee, Woo-Young;Park, Sun-Hee;Kim, Hae-Yeong
    • Food Science and Biotechnology
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    • v.17 no.4
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    • pp.829-832
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    • 2008
  • A multiplex polymerase chain reaction (PCR) method was developed to simultaneously detect 4 varieties of genetically modified (GM) cotton. The event-specific primers were used to distinguish the 4 varieties of GM cotton (MON1445, MON15985, MON88913, and LLcotton25) using multiplex PCR. The acyl carrier protein 1 (Acp1) gene was used as an endogenous reference gene of cotton in the PCR detection. The primer pair Acp1-AF/AR containing a 99 bp amplicon was used to amplify the Acp1 gene and no amplified product was observed in any of the 13 different plants used as templates. This multiplex PCR method allowed for the detection of event-specific targets in a genomic DNA mixture of up to 1% GM cotton containing MON1445, MON15985, MON88913, and LLcotton25.

Detection of Transgenic Rice Containing CrylAc Gene Derived from Bacillus thuringiensis by PCR

  • Kim, Jae-Hwan;Jee, Sang-Mi;Park, Cheon-Seok;Kim, Hae-Yeong
    • Food Science and Biotechnology
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    • v.15 no.4
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    • pp.625-630
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    • 2006
  • Polymerase chain reaction (PCR) method was developed for the specific detection of insect-resistant rice containing cry1Ac gene derived from Bacillus thuringiensis (Bt). Primers were designed from the 35S promoter, NOS terminator, cry1Ac gene, and sucrose phosphate synthase (SPS) for general screening of Bt rice. By sequencing the PCR products from the two putative kinds of Bt rice, we designed a specific primer from the junction region between the cry1Ac gene and the NOS terminator that had been inserted into Bt rice. The construct-specific primer was employed to amplify a 147 bp product in the two lines of Bt rice. No amplified products were observed from the other Bt crops with various Bt genes introduced. In qualitative PCR analysis, the limit of detection was 0.005 ng from genomic DNA of Bt rice. In addition, PCR analysis was performed on 64 kinds of rice presently available in the Korean market, and no Bt rice was detected. This method presented in this paper can be used as a highly sensitive and specific detection method of Bt rice.