• Reproductive and Developmental Biology
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• 제29권4호
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• pp.213-221
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• 2005

We used nine decamer primers to generate DNA fragment sizes ranging from 100 bp to 1,600 bp from two bullhead (Pseudobagrus fulvidraco) populations of Dangjin in Korea. 376 fragments were identified in the cultured bullhead population, and 454 in the population of wild bullhead from Dangjin: 287 specific fragments $(76.3\%)$ in the cultured bullhead population and 207 $(45.6\%)$ in the wild bullhead population. On average, a decamer primer was used to generate 34.2 amplified products in a cultured bullhead. A RAPD primer was used to generate an average of 3.1 amplified bands per sample, ranging between 2.5 and 6.0 fragments in this population. Nine primers also generated 24 polymorphic fragments (24/376 fragment, $6.4\%$) in the cultured bullhead population, and 24 (24/454 fragments, $5.2\%$) in the wild bullhead population. The OPA-16 primer, notably, produced which 11 out of 11 bands $(100\%)$ were monomorphic in the wild bullhead population. 110 intra-population-specific fragments, with an average of 12.2 per primer, were observed in the cultured bullhead population. 99 fragments, with an average of 11.0 per primer, were identified in the wild bullhead. Especially, 55 inter-population-common fragments, with an average of 6.1 per primer, were observed in the two bullhead populations. The bandsharing value (BS value) of individuals within the wild bullhead population was substantially higher than was determined in the cultured bullhead population. The average bandsharing value was $0.596\pm0.010$ within the cultured bullhead population,. and $0.657\pm0.010$ within the wild bullhead population. The dendrogram obtained with the nine primers indicates two genetic clusters, designated cluster $1\;(CULTURED\;01\~CULTURED\;11)$, and cluster $2\;(WILD\;12\~WILD\;22)$. Ultimately, the longest genetic distance displaying significant molecular differences was determined to exist between individuals in the two bullhead populations, namely between individuals WILD no. 19 of the wild bullhead population and CULTURED no. 03 of the cultured bullhead population (genetic distance = 0.714). RAPD-PCR allowed us to detect the existence of population discrimination and genetic variation in Korean population of bullhead. This finding indicates that this method constitutes a suitable tool for DNA comparison, both within and between individuals, populations, species, and genera.

• 한국가축번식학회지
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• 제20권2호
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• pp.89-99
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• 1996

The purpose of this study was to develop the diagnosis techniques for sex determination of rabbit embryos at preimplantation stage. To detect male specific sequences using polymerase chain reaction, two genes functional on sex determination including SRY and ZFX/Y genes were targeted using multiple oligonucleotide primer sets. Three of them for conserved SRY gene were used for appropriate amplification pattern, and then only one primer set #3 proved to be most efficient, showing male-specific strong signal ofamplified sequences. Using this male specific bandsfrom human, cattle, pig and mouse, the gender of rabbit was determined. As an another system for sex determination system, amplified 910bp fragment from ZFX/Y was digested with several restriction endonuclease and showed gender specific restriction fragments only by Hinf I. Using two different system for sex identification of rabbit in this study, blind tests for 17 samples was conducted and showed identical results from two different methods. And then, amplification limit of PCR reaction for template DNA was estimated using various amounts of DNA for both SRY and ZFX/Y systems, resulted as 20pg and 800pg, respectively. With this results, test for gender identification of rabbit embryos were performed using SRY derived amplification system. From total 22 embryos selected for its developmental state 18 were identified as male embryos, showing significant difference from expected sex ratio 1:1. This biased sex ratio was interpreted as to have been caused by the fact, reported by the fact, reported by several researchers, that male embryos develop more rapidly and are more resistant against the in vitro manipulation than female embryos.

• BMB Reports
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• 제37권2호
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• pp.213-222
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• 2004

A randomly amplified polymorphic DNA (RAPD) analysis was used to identify the species- and population-specific markers of abalone; Haliotis asinina, H. ovina, and H. varia in Thai waters. Fifteen species-specific and six population-specific RAPD markers were identified. In addition, an 1650 bp band (UBC195) that was restricted to H. ovina from the Gulf of Thailand (east) was also found. All of the specific RAPD markers were cloned and sequenced. Twenty pairs of primers were designed and specificity-tested (N = 12 and 4 for target and non-target species, respectively). Seven primer pairs (CUHA1, 2, 4, 11, 12, 13, and 14) were specifically amplified by H. asinina DNA, whereas a single pair of primers showed specificity with H. ovina (CUHO3) and H. varia (CUHV1), respectively. Four primer pairs, including CUHA2, CUHA12, CUHO3, and CUHV1, were further examined against 216 individuals of abalone (N = 111, 73, and 32, respectively). Results indicated the species-specific nature of all of them, except CUHO3, with the sensitivity of detection of 100 pg and 20 pg of the target DNA template for CUHA2 and CUHA12 and CUHV1, respectively. The species-origin of the frozen, ethanol-preserved, dried, and boiled H. asinina specimens could also be successfully identified by CUHA2.

• 한국발생생물학회지:발생과생식
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• 제10권4호
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• pp.227-238
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• 2006

한반도의 동쪽에 위치해 있는 삼척(venus clam from Samcheok; VCS)과 원산(venus clam from Wonsan; VCW) 지역에서 채취된 민들조개(Gomphina aequilatera)에서 genomic DNAs(gDNAs)를 분리 추출하였다. 증폭산물은 primer agarose 전기영동법에 의해서 생성되었고, EtBr에 의해서 염색된 이후에 자외선에 의해서 확인되었다. 150 bp에서 2,400 bp에 해당되는 shared loci, polymorphic 및 specific loci를 얻기 위해서 BION-21, BION-23, BION-25, BION-27, BION-29, BION-31 및 BION-33와 같은 7개의 primer를 사용하였다. 본 연구에서 7개의 primer는 VCS 민들조개 집단에서 147개의 polymorphic loci(147/954 loci, 15.41%)와 VCW 집단에서 274개의 polymorphic loci(274/996 loci, 27.51%)를 확인하였다. 이것은 VCS 민들조개 집단에서 보다 VCW 집단에서 더 높은 유전적 변이를 나타내고 있다는 것을 제시하고 있다. 특히 BION-21 primer에 의해서 나타난 700 bp는 민들조개 2개 집단에서 공통적으로 확인되었으며, 이러한 것은 집단이나 종을 확인할 수 있는 marker로서 활용이 가능할 것이다. 이러한 특이한 primer는 개체, 종 및 집단에서 서로 다른 DNA 다형성을 나타내며, 개체나 집단을 확인하는 데 유용하다는 것을 알 수 있다. 2개 민들조개 집단의 개체들을 비교해 보았을 때 SAMCHEOK no. 03와 WONSAN no. 22에서 가장 긴 유전적 거리(0.696)를 나타내었다. 3개의 genetic groupings and dendrogram을 포함한 complete linkage cluster analysis을 통해서 볼 때 지리적 거리가 있었지만 삼척과 원산 2 민들조개 집단의 개체 정체성과 다소 가까운 친척관계를 확인시켜 주었다. 분자적인 표지인자로부터 얻어진 종내 분류와 clustering analyses은 패각 크기, 패각 형태 및 패각 색깔과 같은 형태적인 형질을 기초한 재래적인 종 분류를 지원하고 있다. 따라서 위에서 언급된 바와 같이 RAPD 분석은 VCS 민들조개 집단이 VCW 집단과 어느 정도 차이가 있다는 것을 확인시켜 주었다.

• 대한의생명과학회지
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• 제2권2호
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• pp.241-247
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• 1996

G+C 함량이 높은 primer를 이용한 중합효소 연쇄반응을 실시하는 경우 높은 annealing 온도로 인하여 특이 염기서열의 합성정도가 매우 미약하게 나타나는 경우가 많이 있다. 이와 같은 문제점을 보완하기 위하여 glycerol, formamide 및 dimethyl sulfloxide (DMSO) 등의 변성제를 반응용완충액에 첨가하고 중합효소 연쇄반응을 실시하여 그 결과를 비교 검토하였다. G+C 함량이 낮은 Borrelia burgdorferi의 Lyl 유전자의 primer set인 Bb 679와 Bb 680를 이용한 중합효소 연쇄반응에서는 변성체 첨가에 따른 합성 DNA의 양의 변화가 뚜렷하지 않았다. 그러나 G+C 함량이 높은 primer set인 Mycobacterium paratuberculosis의 IS900 유전자의 IS900/150C와 IS900/921를 이용한 중합효소 연쇄반응을 유도한 경우에는 변성제를 첨가함에 따라 합성된 DNA의 양의 증가가 뚜렷하였으며, 2.5％ glycerol과 1.25％ formamide를 혼합 첨가한 경우와 또는 2.5％ DMSO를 반응용 완충액에 첨가하였을 때 비특이적인 증폭비율이 낮아 특이 염기서열의 합성 결과가 양호한 것으로 나타났다.

• 한국작물학회지
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• 제49권4호
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• pp.358-362
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• 2004

In order to develop molecular markers related to stay green phenotype, AFLP analysis was conducted using near-isogenic lines for either stay green or non stay green trait. Both lines have characteristics of multi-ear and tillers (MET). Two out of 64 primer combinations of selective amplification identified three reproducible polymorphic fragments in MET corn with stay green. Both of E+AGC/M+CAC and E+AAG/M+CAA primer combinations produced two and one specific polymorphic fragments linked to stay green trait, respectively. For the conversion of AFLPs to sequence tag sites (STSs), primers were designed form both end sequences of each two polymorphic fragments. One fragment, which was amplified with E+AAG/M+CAA primer combinations, possessed 298 bp long and showed a $91\%$ homology with maize retrotransposon Cinful-l. One out of two polymorphic fragments produced with E+AGC/M+CAC primer combination had 236 bp long and matched a $96\%$ homology with an intron region of 22kDa alpha zein gene cluster in Zea mays. One out of two PCR fragments amplified with MET2 primer set in the stay green MET was not produced in the non-stay green MET. The developed AFLP and STS marker could be used as an efficient tool for selection of the stay green trait in the MET inbred.

• 한국패류학회지
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• 제22권2호
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• pp.97-108
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• 2006

Genomic DNA samples isolated from geographical purple Washington clam (Saxidomus purpuratus) were obtained from two different regions in Korean Peninsula: Gunsan (Gunsan population; GSP), and Haeju (Haeju population; HJP), a collection area in the vicinity of the West Sea. The seven arbitrarily primers, OPA-07, OPA-09, OPA-18, OPA-20, OPC-03, OPC-06 and OPC-09 were shown to generate the total loci, loci observed per primer, shared loci by each population, specific, and polymorphic loci which could be clearly scored. We also generated the unique shared loci to each population and shared loci by the two populations in purple Washington clam. The size of the DNA fragments also varied wildly, from 50 to 2,400 bp. Here, 304 total loci were identified in the GSP purple Washington clam population, and 282 in the HJP: 91 polymorphic loci (29.9%) in the GSP and 47 (16.7) in the HJP. 198 shared loci, with an average of 28.3 per primer, were observed in the GSP population. The decamer primer OPA-07 generated the shared loci by the two populations, approximately 1,000 bp, between the two Saxidomus populations. The oligonucleotide primer OPC-03 also generated the shared loci by the two populations, approximately 500 bp and 1,000 bp, in GSP population from Gunsan and HJP population from Haeju. The other primer, OPC-06 generated the shared loci by two Gomphina populations (approximately 400 bp). The dendrogram, generated by seven reliable primers, indicates three genetic clusters. The dendrogram obtained by the seven primers indicates three genetic clusters: cluster 1 (GUNSAN 01-GUNSAN 02), cluster 2 (GUNSAN 03-GUNSAN 11), and cluster 3 (HAEJU 12-HAEJU 22). The genetic distance between the two geographical populations ranged from 0.043 to 0.506. Especially, the longest genetic distance displaying significant molecular differences, 0.506, was found to exist between individuals GUNSAN no. 11 of Gunsan and HAEJU no. 17 of Haeju.

• 한국식품과학회지
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• 제31권6호
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• pp.1628-1634
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• 1999

Listeria monocytogenes의 식품 속에서 신속하고 특이적인 검출을 위하여, listeriolysin O gene에 의한 primer, LM 1과 LM 2를 선택하여 PCR을 수행하였다. L. monocytogenes의 DNA 추출이나 cell lysis 없이 intact whole cell을 직접 이용하여 PCR을 하였으며 $10^{2-6}$ CFU 수준의 균체 배양액으로부터 L. monocytogenes에 특이적인 702 bp의 PCR 증폭 산물을 확인하였다. 우유, 닭고기, 김치 등의 식품에 L. monocytogenes를 접종하여 증균배양 전후 균의 PCR에 대한 감도와 생균수를 비교한 결과, 실험된 식품 속에서의 L. monocytogenes의 검출 감도는 순수 배양액에서의 경우에 비해 약 1/10로 둔화되었으나 역시 특이적인 검출이 가능하였다. Primer LM 1과 2를 이용한 본 실험조건에서의 L. monocytogenes의 PCR에 의한 검출은 약 4시간으로 확인이 가능하였으며, 기존의 배양 방법에 비해, 특이성이나 검출 속도 면에서 식품위생 실무에 적용하기 위한 높은 잠재력을 보여 주었다.

• The Plant Pathology Journal
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• 제39권5호
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• pp.494-503
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• 2023

Xanthomonas campestris pv. campestris (Xcc) is a plant pathogen of Brassica crops that causes black rot disease throughout the world. At present, 11 physiological races of Xcc (races 1-11) have been reported. The conventional method of using differential cultivars for Xcc race detection is not accurate and it is laborious and time-consuming. Therefore, the development of specific molecular markers has been used as a substitute tool because it offers an accurate and reliable result, particularly a quick diagnosis of Xcc races. Previously, our laboratory has successfully developed race-specific molecular markers for Xcc races 1-6. In this study, specific molecular markers to identify Xcc race 7 have been developed. In the course of study, whole genome sequences of several Xcc races, X. campestris pv. incanae, X. campestris pv. raphani, and X. campestris pv. vesicatoria were aligned to identify variable regions like sequence-characterized amplified regions and insertions and deletions specific to race 7. Primer pairs were designed targeting these regions and validated against 22 samples. The polymerase chain reaction analysis revealed that three primer pairs specifically amplified the DNA fragment corresponding to race 7. The obtained finding clearly demonstrates the efficiency of the newly developed markers in accurately detecting Xcc race 7 among the other races. These results indicated that the newly developed marker can successfully and rapidly detect Xcc race 7 from other races. This study represents the first report on the successful development of specific molecular markers for Xcc race 7.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.96-96
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• 2002

상동유전자 재조합기술을 myostatin 유전자에 적용하기 위해 돼지 골격에 붙어 있는 근육으로부터 RNA를 추출하였고 돼지 Myostain Exon 3 부위의 specific primer를 제작하여 RT-PCR 을 수행 한 후 증폭된 342bp DNA 를 추출하여 T vector 에 ligation한 후 sequencing을 실시하여 돼지 genomic DNA 에서 Myostatin gene 의 Exon 3 부위와 100% match 되는 것을 확인하였다. (omitted)