• Journal of Microbiology and Biotechnology
• /
• 제21권12호
• /
• pp.1336-1344
• /
• 2011

This study details and examines a novel ligation-mediated polymerase chain reaction (LM-PCR) method. Named the LM-PCR/Shifter, it relies on the use of a Class IIS restriction enzyme giving restriction fragments with different 4-base, 5' overhangs, this being the Shifter, and the ligation of appropriate oligonucleotide adapters. A sequence of 4-base, 5' overhangs of the adapter and a 4-base sequence of the 3' end of the primer(s) determine a subset of the genomic restriction fragments, which are amplified by PCR. The method permits the differentiation of bacterial species strains on the basis of the different DNA band patterns obtained after electrophoresis in polyacrylamide gels stained with ethidium bromide and visualized in UV light. The usefulness of the LM-PCR/Shifter method for genotyping is analyzed by a comparison with the restriction endonuclease analysis of chromosomal DNA by the pulsed-field gel electrophoresis (REA-PFGE) and PCR melting profile (PCR MP) methods for isolates of clinical origin. The clustering of the LM-PCR/Shifter fingerprinting data matched those of the REA-PFGE and PCR MP methods. We found that the LM-PCR/Shifter is rapid, and offers good discriminatory power and excellent reproducibility, making it a method that may be effectively applied in epidemiological studies.

• Asian-Australasian Journal of Animal Sciences
• /
• 제18권9호
• /
• pp.1226-1230
• /
• 2005

The present investigation was undertaken to study the genetic polymorphism of the DRB3 exon 2 in 75 crossbred cattle by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Five genotypes i.e. HaeIII-a, HaeIII-b, HaeIII-e, HaeIII-ab and HaeIII-ae were observed when the 284 bp PCR products were digested with HaeIII restriction enzyme. The corresponding frequencies of these patterns were 0.53, 0.04, 0.01, 0.38 and 0.04, respectively. Digestion with RsaI restriction enzyme resolved 24 different restriction patterns. The frequencies of these patterns ranged from 0.013 (RsaI-f, RsaI-k and RsaI-c/n) to 0.120 (RsaI-n). The results revealed that the crossbred cows belonged to the RsaI patterns namely b, k, l, a/l, d/s, l/n, l/o and m/n, whose corresponding frequencies were 0.027, 0.013, 0.040, 0.027, 0.040, 0.067, 0.027 and 0.067, respectively. Digestion of the 284 bp PCR product of DRB3.2 gene with PstI in the crossbred cattle did not reveal any restriction site. These results suggested the absence of the recognition site in some of the animals. These results also revealed that the crossbred cows studied were in homozygous as well as heterozygous condition. On the basis of the above results it can be concluded that the DRB3.2 gene was found to be highly polymorphic in the crossbred cattle population.

• 한국동물학회:학술대회논문집
• /
• 한국동물학회 1994년도 한국생물과학협회 학술발표대회
• /
• pp.90.2-90
• /
• 1994

No Abstract, See Full Text

• 미생물학회지
• /
• 제32권4호
• /
• pp.245-251
• /
• 1994

영지속(Ganoderma)에 속하는 7종 10균주에 대하여 미토콘드리아 DNA의 제한효소 분절양상 비교를 통한 계통분석을 수행하였다. 여러 가지 제한효소들 중 생산된 절편이 충분한 정보를 가지고 있으면서 서로 구별할 수 있는 6가지의 제한효소를 분석에 이용하였다. 절편양상을 설 비교하여 전체 절편중 공통된 절편의 개수를 구하고 이로부터 염기위치당 염기치환율을 구하였으며, 이를 균주간의 진화거리로 계산하여 PHYLIP package의 Neighbor-joining 방법에 이한 계통도를 얻고 그 결과를 고찰하였다. 특이한점은 G. lucidum의 3균주와 G. lobatum 이 유연관계가 많이 있다는 점이다. 이러한 결과는 G. lucidum과 G. lobatum은 종의 다양성으로 인하여 과거부터 복합종으로 취급되어 왔으며 고전적인 영지속의 분류에 문제점이 많이 있음을 시사해 주고 있다. 따라서 영지속의 분류가 진화경로에 바탕을 둔 자연분류가 되기 위해서는 형태분류 뿐만 아니라 배양 분류와 분자생물학적이 sqnstjr등 다양한 기준에 의해서 재고되어야 할 것으로 판단된다.

• Clinical and Experimental Pediatrics
• /
• 제49권11호
• /
• pp.1152-1157
• /
• 2006

Inherited metabolic disease is rare disorders that show symptoms mainly in pediatric age and early treatment is important for preventing complications of the disease. Recent development in molecular and biochemical techniques help clinicians with proper diagnosis of patients, however, many of the disease still remain lack of effective therapeutic strategies. Better understanding on biochemical and molecular basis of pathogenesis of the disease combined with advanced medical care would provide new sight on the disease that can also improve the quality of life and long-term prognosis of patients. Traditionally, there are several modalities in the treatment of metabolic diseases depend on the biochemical basis of the disease such as diet restriction, removing or blocking the production of toxic metabolites, and stimulating residual enzyme activity. The inherited metabolic disease is not familiar for many clinicians because the diagnosis is troublesome, treatment is complicated and prognosis may not as good as expected in other diseases. Recently, new therapeutic regimens have been introduced that can significantly improve the medical care of patients with metabolic disease. Enzyme replacement therapy has showed promising efficacy for lysosomal storage disease, bone marrow transplantation is effective in some disease and gene therapy has been trying for different diseases. The new trials for treatment of the disease will give us promising insight on the disease and most clinicians should have more interest in medical progress of the metabolic disease.

• 미생물학회지
• /
• 제26권1호
• /
• pp.6-12
• /
• 1988

Cellobiase ($\beta$-glucosidase) is an enzyme of the cellulase system in cellulolytic microor-ganisms. The chromosomal DNA fragment which include cellobiase gene of Cellulomonas biazotea was cloned in Eschericia coli via plasmid pBR 322 vector. Restriction enzyme Sal I was used to obtain adequate size of fragments from C. biazotea. chromosomal DNA. The transformant of E. coli HB101 with recombinant plasmid pBG101 showed cellobiase activity, which is not ordinary in E. coli HB101. The enzyme activity of the transformant was as of 20% lower than that of C. biazotea.

• Asian-Australasian Journal of Animal Sciences
• /
• 제27권10호
• /
• pp.1487-1492
• /
• 2014

This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp). Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment.

• Parasites, Hosts and Diseases
• /
• 제55권6호
• /
• pp.653-658
• /
• 2017

Hypoderma spp. larvae cause subcutaneous myiasis in several animal species. The objective of the present investigation was to identify and characterize morphologically and molecularly the larvae of Hypoderma spp. collected from cattle (Bos taurus taurus) and red deer (Cervus elaphus) in the district of Castelo Branco, Portugal. For this purpose, a total of 8 larvae were collected from cattle (n=2) and red deer (n=6). After morphological identification of Hypoderma spp. larvae, molecular characterization was based on PCR-RFLP and mitochondrial CO1 gene sequence analysis. All larvae were morphologically characterized as the third instar larvae (L3) of H. actaeon. Two restriction enzymes were used for molecular identification of the larvae. TaqI restriction enzyme was not able to cut H. actaeon. However, MboII restriction enzyme differentiated Hypoderma species showing 210 and 450 bp bands in H. actaeon. Furthermore, according to the alignment of the mt-CO1 gene sequences of Hypoderma species and to PCR-RFLP findings, all the identified Hypoderma larvae were confirmed as H. actaeon. This is the first report of identification of Hypoderma spp. (Diptera; Oestridae) from cattle and red deer in Portugal, based on morphological and molecular analyses.

• 한국미생물·생명공학회지
• /
• 제15권5호
• /
• pp.352-355
• /
• 1987

새로운 제한효소인 Cth I을 Clostridium thermocellum ATCC 27405로부터 분리하여, 그 효소의 생화학적 특성을 연구하였다. 이 효소는 Bcl I endonuclease의 isoschizomer로서 5'-TGATCA-3'를 인식한다. Cth I endonuclease는 섭씨 60도의 높은 온도에서 잘 작용하며, 10mM까지의 저농도의 NaCl과 MG$^{2+}$ 이온의 존재하에서 pH7.5와 10.5사이에서 최적의 활성을 보여주었다. Cth I endonuclease의 활성은 DNA 기질이 dam methylation 되었을 때 저해를 받았다.

• 한국작물학회지
• /
• 제46권1호
• /
• pp.53-56
• /
• 2001

Barley Yellow Dwarf Virus (BYDV), an aphid-borne luteovirus, is a major plant pathogenic disease causing a huge economic loss in the grain production of a wide range of Gramineae species throughout the world. It has been recently reported that BYDV also occurred frequently in wheat field of Korea. Here, we performed to develop the detection and classification methods of BYDV strains that were accomplished by reverse transcription-polymerase chain reaction (RT-PCR). Since there are high variations among BYDV strains, three pairs of primers were designed to detect BYDV strains such as PAV (Vic-PAV and CN-PAV) and MAV (primer A) simultaneously, specifically Vic-PAV(primer B), and MAV (primer C) based on the genomic RNA sequences of BYDV strains previously published. The validity of the primers was confirmed using several BYDV strains obtained from CIMMYT. Though three BYDV strains were able to be detected using primer A, PCR products were not distinguished between two PAV strains. It was possible to separate them with a restriction enzyme, EcoRI, whose restriction site was present in the amplified DNA fragment from Vic-PAV, but not from CN-PAV.