• Title, Summary, Keyword: AP-PCR

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A Genetic Marker for the Korean Native Cattle (Hanwoo) Found by an Arbitrarily Primed-Polymerase Chain Reaction (AP-PCR)

  • Lee, Ji-Seon;Lee, Chang-Hee;Nam, Doo-Hyun;Jung, Young-Ja;Yeo, Jung-Sou
    • BMB Reports
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    • v.33 no.3
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    • pp.208-212
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    • 2000
  • In order to develop a specific genetic marker for the Korean native cattle (Hanwoo), an arbitrarily-primed polymerase chain reaction (AP-PCR) analysis of 6 different cattle breeds was attempted. Eight different arbitrary primers, each longer than 20-mer nucleotides, were used. In comparison to the AP-PCR patterns, several distinctive DNA bands that are specific for a certain breed were detected. When the primer Kpn-X was employed, a 280bp DNA fragment was found to be specific only for Hanwoo. In an individual analysis of Hanwoo, this AP-PCR marker was observed in 123 head of cattle among the 153 that were tested (80.4%). Nucleotide sequencing revealed that this fragment has a short microsatellite sequence of tandem repeat, $A(G)_{1-2}\;(C)_{1-3}AGAG$. According to the analysis of AP-PCR band patterns, Hanwoo was discovered to be genetically most closely-related with Holstein among the various cattle breeds.

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Epidemiological Investigation of Methicillin-Resistant Staphylococcus aureus by Arbitrarily Primed PCR

  • Yang Byoung-Seon
    • Biomedical Science Letters
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    • v.10 no.4
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    • pp.473-477
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    • 2004
  • Methicillin-Resistant Staphylococcus aureus (MRSA) strains are resistant to a wide range of antibiotics and are a major cause of nosocomial infections. Accurate and rapid typing of MRSA is needed to implement effective infection control measures. Arbitrarily Primed PCR (AP-PCR) is a very useful method in rapid typing. AP-PCR is not necessary information about target DNA sequence because this is basically DNA amplification and could be useful in epidemiological typing by classified band pattern. In this study, MRSA were isolated and identified from ICU, Neu, IM and Ped environments and investigated molecular typing by AP-PCR. Ped, the MRSA pattern determines the la, IIa type, 1M is Ib type, Neu is IIa type and ICU determines the IIa, lIb types. All MRSA in this study were typeable by AP-PCR, which was easy to perform and reproduce with evidence of MRSA for purposes of nosocomial infection control.

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Follow Up Expression Patterns of Alkaline Phosphatase(AP) as a Marker for Establishing Mouse Embryonic Stem (ES) Cells (배아주간세포수립을 위한 Alkaline Phosphatase(AP)의 상이한 발현 양식의 추적)

  • 김진회;차수경;노민경;송상진;구덕본;이훈택;정길생
    • Korean Journal of Animal Reproduction
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    • v.19 no.1
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    • pp.55-63
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    • 1995
  • The putative totipotency germ cells has a relative abundance of alkaline phosphatases. Thus, histological staining of AP activity offers a new route to isolate totipotent cells and also provides insights into culture systems of these cells. Furthermore, the AP staining technique is simple and fast, requires only the napthol AS/MS substrate in combination with trapping diazonium salts such as fast red or fast blue. However, our unexpected finding was that AP staining of mouse ES cells were detected in the undifferentiaed epiblast-derived cells as well as several types of differentiating cells. This findings are different from results of Talbot et al. (1993) reported usefulness of the AP staining and implies that histological staining of AP may not by useful to determine undifferentiaed state or totipotency of ES cells. Thus, we have investigated the patterns of AP expression by RT-PCR in order to identify a marker of undifferentiated ES/primordial germ (PG) cells. In RT-PCR analysis, embryonic (E)-AP was detected only in undifferentiated ES cells, but intestinal(I)-AP was not detected in all of the examined ES and PG cells. In addition, nonspecific (NS)-AP wasdetected in undifferentiated PG cell from day 7, 5 to 13 of gestation. Histological activity of AP in ES cells was completely suppressed by addition of L-phenylalanine (Phe), L-homoarginine (Har), and L-phenylalanylglycylglycine (PheGlyGly) as an inhibitor, but RT-PCR showed the same results as in the absence of an inhibitors. Our findings suggested that expression of E-AP and NS-AP may use as a marker to determine the undifferentiated status in ES and PG cells.

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EVALUATING TWO METHODS FOR FINGERPRINTING GENOMES FOR STREPTOCOCCUS MUTANS IN CHILDREN : A COMPARISON WITH AP-PCR AND SOUTHERN BLOT RFLP (유전자형에 따른 Streptococcus mutans의 subtyping: Southern blot RFLP와 AP-PCR을 이용한 비교)

  • Jeong, Tae-Sung;Kim, Shin
    • JOURNAL OF THE KOREAN ACADEMY OF PEDTATRIC DENTISTRY
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    • v.25 no.2
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    • pp.292-303
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    • 1998
  • The arbitrary primer polymerase chain reaction(AP-PCR) and Southern blot restriction fragment length polymorphism(RFLP) were used to genotype the cariogenic pathogen S. mutans in children. Following the morphologic chracteristics of colony on selective medium for S. mutans, total genomic DNA from 155 strains was extracted by conventional methods. Among 155 strains, 143 strains (92.3%) were confirmed S. mutans by PCR with dexA gene and 114 strains were used in this study. Three random sequence 10-base oligonucleotide primers were chosen for AP-PCR. The amplified DNA products were separated electrophoretically in a 2% agarose gel containing ethidium bromide and the banding patterns were compared among different strains. For RFLP analysis, DNA was digested with EcoRI and BamHI, separated on a 0.7 % agarose gel and transferred to a nylon membrane. The membrane was probed with a previously characterised 1.6 kilobases (kb) DNA fragment cloned from gtf B gene of S. mutans. The probe was labeled with isotope[$^{32}P-{\alpha}CTP$], and hybridized fragments were detected with intensifying screen. AP-PCR produced 4-8 DNA bands in the 0.25-10 kb regions and distinguished 9, 10 or 12 genotypes, depending on the specific primer used. Southern blot RFLP analysis revealed 2 hybridization patterns consisting of 1 DNA fragments 450, 500 bp. These results indicate that AP-PCR is more discriminative method for genotyping of S. mutans.

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Fingerprinting of Rice Genomes Using PCR with Arbitrary Primers

  • Park, Kyong-Hee
    • Preventive Nutrition and Food Science
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    • v.3 no.2
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    • pp.198-202
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    • 1998
  • The arbitrary primed polymerase chain reaction (AP-PCR) has been used to detect the genetic alternations in the related species. Simple and reproducible fingerprints of complex genomes can be generated using single arbitrary chosen primers and the PCR. The technique was applied to the Oryza species and characterized the relationship among three cultivars of rice species based on theresult of genomic DNA fingerprints. The results indicated that the polymorphism revealed in rice strains and the differences in the PCR product pattern could be represented for each strainis. There was many variationsin the PCR product pattern between cv. Dongin(japonica type)and cv.Hyangdo (indica type), and our chosen AP-primers can ge as markers for strain identification and verfication.

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Polymorphism of Salmonella Strains Using Arbitrary-Primed Polymerase Chain Reaction (Arbitrary-Primed PCR 기법을 이용한 Salmonella 균의 다형성 분석)

  • Hwang, Eui-Kyung;Kim, Sang-Kyun;Kim, Yeon-Soo;Kim, Woo-Tea;Lee, Jeong-Koo
    • Korean Journal of Veterinary Research
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    • v.42 no.2
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    • pp.191-199
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    • 2002
  • In this study, eight primers were used to detect genetic variability and phylogenetic relationships among the eighteen Salmonella strains by the arbitrary-primed PCR(AP-PCR) techniques. Five strains of Salmonella typhimurium, four strains of S entertidis, three strains of S choleraeuis, three strains of S gallinarum and three strains of S pullorum were typed by AP-PCR. The number of AP-PCR bands detected per each primer varied from 39 to 52, with an average of 43.6. A total of 349 AP-PCR bands were generated and among them, 185 bands(53.0%) were polymorphic. Among the primers, GEN 703 and GEN 708 primer showed a high level of polymorphism with 0.682 and 0.676, respectively. But GEN 603, GEN 604 and GEN 607 primer showed a low level of polymorphism with 0.404, 0.460 and 0.472, respectively. Therefore, the these primers will be the most effective for AP-PCR analysis of Salmonella strains. The level of polymorphism of S typhimurium CU 2001(0.77) was similar to that of S typhimurium CU 2002(0.77) and lower than those of other strains such as S typhimurium CU 2003(0.63), S typhimurium ATCC 14028(0.50) and S typhimurium CU 2004(0.43). The level of polymorphism of S enteritidis ATCC 13076(0.83) was similar to that of S enteritidis CU 2005(0.83) and lower than those of other strains such as S enteritidis CU 2006(0.63) and S enteritidis CU 2007(0.58). The level of polymorphism of S choleraeuis CU 2009(0.67) was similar to that of S choleraeuis CU 2010(0.67) and higher than those of other strains such as S choleraeuis CU 2008(0.53). The level of polymorphism of S gallinarum CU 2011(0.70) was similar to that of S gallinarum CU 2012(0.70) and higher than those of other strains Such as S gallinarum ATCC 9184(0.60). The level of polymorphism of S pullorum CU 2013(0.80) was similar to that of S pullorum CU 2014(0.80) and higher than those of other strains such as S pullorum No 11(0.53). Therefore, the AP-PCR analysis will be used a powerful tool for estimating genetic variation and phylogenetic relationships among Salmonella strains.

Identification of Varieties by Biochemical Methods in Pleurotus spp. (느타리 버섯류(Pleurotus spp.)의 생화학적 방법에 의한 품종구분)

  • Kim, Dong-Hyun;Kong, Won-Sik;Kim, Kyung-Soo;Kim, Young-Ho;You, Chang-Hyun;Kim, Young-Bae
    • The Korean Journal of Mycology
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    • v.26 no.2
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    • pp.173-181
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    • 1998
  • To identify genetic difference of 13 strains in three Pleurotus species, analyses of rDNA, AP-PCR and RFLP were carried out. IGRI and $ITSI{\sim}II$ regions of rDNA amplified by PCR were about 0.9 and 0.7 kb, respectively. These PCR products were digested with six restriction enzymes to analyse polymorphism. Especially, treatment of HaeIII enzyme on $ITSI{\sim}II$ regions showed specific bands in three Pleurotus sajor-caju strains. Genetic differences among three species were classified by similarity analyses based on rDNA polymorphism. Various band patterns of $2,500{\sim}150\;bp$ were showed by AP-PCR. Identification of species and varieties in 13 Pleurotus strains was possible according to primers used in AP-PCR. In order to develop genetic markers, RFLPs using IGRI and $ITSI{\sim}II$ probes derived from ASI 2180 and 2070 were carried out on eight Pleurotus varieties. RFLP patterns using IGRI probe were more various than that of $ITSI{\sim}II$ probe.

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GENOTYPING OF STREPTOCOCCUS MUTANS USING AP-PCR IN CHILDREN WITH RAMPANT CARIES (AP-PCR을 이용한 다발성 우식아동의 구강내 Streptococcus mutans의 유전자형 분류)

  • Jang, Myung-Jo;Kim, Shin
    • JOURNAL OF THE KOREAN ACADEMY OF PEDTATRIC DENTISTRY
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    • v.24 no.1
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    • pp.65-81
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    • 1997
  • For the purpose of evaluating the appropriateness of AP-PCR as a facile, rapid and reproducible method for genotyping Streptococcus mutans, and selecting the discriminant primer for it, a DNA fingerprinting was performed on the microorganisms isolated from caries-free children and children with rampant caries, respectively. In the course of selecting appropriate primer for S. mutans genotyping, we chose S2 primer from 6 different primers which shows highest resolution on the agarose gel as well. Nineteen kinds of fingerprint patterns were observed in caries-free children and children with rampant caries which were produced by combination of 7 different fragments. Interestingly, the number of types observed in caries-free children was greater than that in children with rampant caries. And we observed Type 2 was predominant in children with rampant caries (about 80%) and relatively even distribution of each types in caries-free children. Furthermore, it was appeared that the major types in normal control were not or rarely found in children with rampant caries. In conclusion, we could establish simple, rapid and highly reproducible AP-PCR method for genotyping S. mutans. We also found differences in distribution of S. mutans between normal and patient, which suggested that cariogenicity is also dependent on qualitative aspects which is caused by the difference in genotypes of S. mutans in oral cavity.

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Genotypic Analysis of Multi-drug Resistant Staphylococcus aureus by Arbitrarily Primed Polymerase Chain Reaction (AP-PCR을 이용한 다제내성 Staphylococcus aureus의 유전형 분석)

  • Shin, Kyoung Hyun;Hong, Seung Bok;Son, Seung Yeol
    • Korean Journal of Clinical Laboratory Science
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    • v.36 no.2
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    • pp.89-97
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    • 2004
  • Many strains of Staphylococcus aureus were isolated from pus samples from primary, secondary, and tertiary medical institutions and were subjected to an antibiotic sensitivity test. Ciprofloxacin, clindamycin, erythromycin, gentamicin, oxacillin penicillin, tetracycline, trimethoprim/sulfamethoxazole, vancomycin and teicoplanin were used for the antibiotic sensitivity test. The strains showed hightest resistance to penicillin(91%), but all of strains tested were susceptible to vancomycin and teicoplanin. The isolated multi-drug(penicillin-tetracycline-ciprofloxacin-clindamycin-erythromycin- oxacillin-gentamicin) resistant S. aureus were analyzed genotypically using an AP-PCR(Arbitrarily Primed polymerase chain reaction) with an arbitrary 3 primers. Based on the result for genotype analysis, the genotypes identified by S1 primer did not coincide with those of S2 or E2 primers. Genotypes identified by S2 primer did not coincide with those of S1 or E2 primers. Also genotypes identified by the E2 primer did not coincide with those of S1 or S2 primers. Therefore, an analysis of AP-PCR test with multiple primers will provide more sensitive identification. A strain from a secondary medical institution and a strain from a tertiary medical institution which showed the same genotype for S1, S2, and E2 primers are required for further epidemiological study.

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Detection of Viruses Infecting Stone Fruits in Western Mediterranean Region of Turkey

  • Yardimci, Bayram Cevik Nejla;Culal-Klllc, Handan
    • The Plant Pathology Journal
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    • v.27 no.1
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    • pp.44-52
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    • 2011
  • Field surveys were conducted in 45 stone fruit orchards in seven districts of Isparta Province located in western Mediterranean region of Turkey important for stone fruit production. Leaf samples were collected from 175 trees showing virus-like symptoms. These samples were first tested by ELISA for five different RNA viruses including Apple mosaic ilarvirus (ApMV), Prunus necrotic ringspot ilarvirus (PNRSV), Prune dwarf ilarvirus (PDV), Plum pox potyvirus (PPV), Apple chlorotic leafspot trichovirus (ACLSV). While no ApMV and PPV infection was found, 46, 24 and 16 samples were tested positive for PDV, ACLSV and PNRSV, respectively, in ELISA showing about 45% of symptomatic trees in the region were infected with at least one of these viruses. In addition, it was found that nine sweet cherry trees were mixed infected with two or three of these viruses and PDV with an infection rate of 26.3% was the most widespread virus in symptomatic trees in western Mediterranean region. Thirty samples were selected and tested by a multiplex RT-PCR (mRT-PCR) for simultaneous detection of these viruses. While PPV was not detected, more than half of the tested 20 samples were individually or mixed infected with ApMV, ACLSV, PNRSV and PDV. The mRT-PCR results were confirmed by detection of these viruses individually in some of the field samples using RT-PCR with primes specific to each virus. Comparison of ELSA and mRT-PCR results of 30 samples showed that numbers of infected and mixed infected samples as well as infection and mixed infection rates were significantly higher in RT-PCR (20 and 66.7%) than in ELISA (14 and 46.7%). The results confirm that mRT-PCR is more sensitive than ELISA.