Type-specific Amplification of 5S rRNA from Panax ginseng Cultivars Using Touchdown (TD) PCR and Direct Sequencing

Sun, Hun,Wang, Hong-Tao,Kwon, Woo-Saeng,Kim, Yeon-Ju,Yang, Deok-Chun The Korean Society of Ginseng 2009 Journal of Ginseng Research Vol.33 No.1

Generally, the direct sequencing through PCR is faster, easier, cheaper, and more practical than clone sequencing. Frequently, standard PCR amplification is usually interpreted by mispriming internal or external regions of the target template. Normally, DNA fragments were eluted from the gel using Gel extraction kit and subjected to direct sequencing or cloning sequencing. Cloning sequencing has often troublesome and needs more time to analyze for many samples. Since touchdown (TD) PCR can generate sufficient and highly specific amplification, it reduces unwanted amplicon generation. Accordingly, TD PCR is a good method for direct sequencing due to amplifying wanted fragment. In plants the 5S-rRNA gene is separated by simple spacers. The 5S-rRNA gene sequence is very well-conserved between plant species while the spacer is species-specific. Therefore, the sequence has been used for phylogenetic studies and species identification. But frequent occurrences of spurious bands caused by complex genomes are encountered in the product spectrum of standard PCR amplification. In conclusion, the TD PCR method can be applied easily to amplify main 5S-rRNA and direct sequencing of panax ginseng cultivars.

Type-specific Amplification of 5S rRNA from Panax ginseng Cultivars Using Touchdown (TD) PCR and Direct Sequencing

Hun Sun,Hong-Tao Wang,Woo-Saeng Kwon,Yeon-Ju Kim,Deok-Chun Yang 고려인삼학회 2009 Journal of Ginseng Research Vol.33 No.1

Generally, the direct sequencing through PCR is faster, easier, cheaper, and more practical than clone sequencing. Frequently, standard PCR amplification is usually interpreted by mispriming internal or external regions of the target template. Normally, DNA fragments were eluted from the gel using Gel extraction kit and subjected to direct sequencing or cloning sequencing. Cloning sequencing has often troublesome and needs more time to analyze for many samples. Since touchdown (TD) PCR can generate sufficient and highly specific amplification, it reduces unwanted amplicon generation. Accordingly, TD PCR is a good method for direct sequencing due to amplifying wanted fragment. In plants the 5S-rRNA gene is separated by simple spacers. The 5S-rRNA gene sequence is very well-conserved between plant species while the spacer is species-specific. Therefore, the sequence has been used for phylogenetic studies and species identification. But frequent occurrences of spurious bands caused by complex genomes are encountered in the product spectrum of standard PCR amplification. In conclusion, the TD PCR method can be applied easily to amplify main 5S-rRNA and direct sequencing of panax ginseng cultivars.

Detection of Lamivudine-Resistant Mutations of HBV DNA Polymerase Gene Using PCR-Direct Sequencing

Lee, Kyung-Ok,Lee, Hye-Jung,Byun, Ji-Young,Lee, Sung-Yeun,Kim, Jeong-Sook,Jung, Na-Young,Chung, Soo-Jin,Seong, Hye-Soon,Kim, Kyung-Tae Korean Society for Clinical Laboratory Science 2006 대한임상검사과학회지(KJCLS) Vol.38 No.3

최근 만성 B형간염의 치료에 B형간염 바이러스 복제를 저해하여 감염력을 약화시키는 lamivudine이 많이 사용되고 있다. 그러나 이 약제를 장기간 복용할 경우 B형간염 바이러스 DNA polymerase 유전자의 YMDD motif에 아미노산 치환을 일으켜 lamivudine 저항성 B형간염 바이러스가 나타나는 점이 문제시 되고 있다. 본 연구의 목적은 lamivudine 치료를 받은 만성 B형간염 환자에서 PCR-direct sequencing 법을 이용하여 B형간염 바이러스 DNA 중합효소 유전자의 YMDD motif(codon 552)와 codon 528에서 돌연변이 출현빈도를 구하고, HBeAg과의 관련성을 보고자 하였다. 방법은 만성 B형간염 환자의 혈청에서 DNA를 추출하고 DNA 중합효소 유전자의 codon 552와 528을 포함하는 부위에서 선택한 두 쌍의 primer를 이용하여 nested PCR을 실시하였다. 증폭된 PCR 산물은 2% agarose gel에서 전기영동을 한 후, 자동염기서열분석기를 이용하여 sequencing을 실시하였다. 총 207명 중에서 돌연변이는 124명(60%)에서 발견되었으며, 남, 녀에서 차이는 발견되지 않았고, 돌연변이군에서 비돌연변이군에 비해 평균나이가 약간 높게 나타났으나 유의성은 없었다. Codon 552에서 단일돌연변이로는 M552I(50.8%)가 가장 높게 나타났고, 다음으로 M552V(43.5%), M552I/V(5.7%)의 순서로 나타났다. Codon 528에서는 67.0%의 L528M 돌연변이가 발견되었다. Codon 552와 codon 528에서 동시에 발생한 중복돌연변이로는 M552V/L528M(43.6%)이 가장 높게 나타났고, 다음으로 M552I/L528(33.1%) 그리고 M552I/L528M(17.7%)의 순으로 나타났다. Codon 552에서 serine 돌연변이(M528S)는 발견되지 않았으며, L528M은 M552V 돌연변이와 거의 동시에 검출되었다. 본 연구에서 만성 B형간염환자에서 HBeAg의 유무와 lamivudine 돌연변이율과의 상관성은 발견되지 않았으며, PCR-direct sequencing법은 고가의 자동염기서열분석 장비와 숙련된 기술자가 필요하다는 문제점은 있으나, 검체 수가 많은 큰 임상검사실에서는 활용성이 클 것으로 판단된다. 향 후 lamivudine으로 인한 HBV 돌연변이형과 환자의 임상결과의 관련성에 대한 연구가 추가적으로 실시되면, lamivudine을 복용하는 만성 HBV 환자의 치료와 예후에 유용할 것으로 사료된다. Treatment of hepatitis B virus (HBV) with lamivudine is effective in suppressing virus replication and results in reduced inflammatory activity. However the most troublesome problem of lamivudine treatment is the emergence of lamivudine-resistant strains with amino acid substitution in the YMDD motif of DNA polymerase gene during the treatment. The aim of this study was to determine the mutation of YMDD motif (codon 552) and codon 528 in chronic HBV patients with lamivudine therapy using PCR-direct sequencing and to investigate the relationship between lamivudine mediated HBV mutation and HBeAg. HBV DNA was extracted from serum samples of HBV patients and amplified by nested PCR with two sets of primer pairs selected in HBV DNA polymerase gene. Amplified PCR product was analyzed by 2% agarose gel electrophoresis and direct sequencing. HBV mutation was detected in 124 out of 207 samples (60%). Single mutation was 50.8% for M552I, 43.5% for M552V, 5.7% for M552I/V and the L528M mutation was 67.0%. Double mutation was 43.6% for M552V/L528M, 33.1% for M552I/L528(wild type), 17.7% for M552I/L528M and 5.6% for M552I/V/L528M. Serine mutation at YMDD motif (M552S) was not found and the L528M mutation frequently accompanied M552V type. In this study, the typical difference of frequencies for HBV mutation depending on HBeAg was not found. Moreover, the PCR-direct sequencing method used in this study might be a powerful tool for the mutation study in clinical reference laboratories with high volume.

PCRㆍ직접염기서열분석법에 의한 ABO 유전형검사 6년 경험

원은정,조덕,허민석,박혜련,신명근,양동욱 대한수혈학회 2012 大韓輸血學會誌 Vol.23 No.3

Background: ABO genotyping is essential for resolving ABO grouping discrepancy and for determinating ABO subgroups. Most clinical samples, including suspected inherited subgroups and acquired variant phenotypes, can be determined by PCR-sequencing of exons 6 and 7 in the ABO gene. Here, we describe our six years' experience performing ABO genotyping by PCR-direct sequencing. Methods: We conducted a retrospective investigation of serological and genotypical data from 205 samples (158patients and 47 of their family members) of patients who were referred to the Molecular Genetics Laboratory at Chonnam National University Hwasun Hospital for ABO genotyping between January 2007 and July 2012. ABO genotyping was performed on all samples with PCR-direct sequencing of exons 6 and 7 in the ABO gene; the standard serologic tests were also performed. Results: The frequency of phenotypes consistent with their genotypes was 70.8% (112/158 cases) and the A2B3phenotype with the cis-AB01 allele was the most common (31.0%, 49 cases) among them. The frequency of phenotypes inconsistent with their genotypes was 29.1% (46/158 cases) and the A1B3 phenotype was the most frequently recovered case (5.1%, 8 cases). Family study showed differential phenotype expression depending on the co-inherited ABO allele in five families with the B306, cis-AB01, Ael02, Aw14, or B305 allele and also showed a typical inheritance of a chimera with A102/B101/O04. Conclusion: We propose that ABO genotyping using PCR-direct sequencing is useful for the resolution of ABO discrepancies and for the investigation of ABO subgroups based on six years' experience. In addition, family study for analysis of phenotypic patterns of ABO subgroups is also crucial to ABO genotyping. 배경: ABO 유전형 검사는 ABO 불일치의 해결및 ABO 아형의 결정에 있어서 매우 중요하다. 대부분 ABO 아형 및 변이 표현형을 보이는 임상검체에서 그 원인은 ABO 유전자의 엑손 6과 7의PCR증폭 및 직접염기서열분석을 통해 결정될 수있다. 본 저자들은 PCR증폭 및 직접염기서열분석을 통한 ABO 유전형 검사를 시행한 6년 간의경험을 소개하고자 한다. 방법: 본 연구는 2007년 1월부터 2012년 7월 사이에 화순전남대학교병원에 의뢰된 205 검체(환자 158명, 가족 47명의 검체)의 ABO 유전자검사결과를 후향적인 분석을 통하여 실시하였다. ABO 유전형 검사는 ABO 유전자의 엑손 6과 7에 대한PCR 증폭 후 직접염기서열분석을 통해 실시하였다. 표준혈청학적 검사 또한 시행되었다. 결과: ABO 유전형 검사가 의뢰된 총 158 검체중 112건(70.8%)은 ‘표현형과 유전형이 일치한 경우’였으며, cis-AB01 대립유전자를 동반하는 A2B3형이 49건(31.0%)으로 가장 흔했다. 표현형과 유전형이 일치하지 않은 경우는 158건 중 46건(29.1%)이었으며, 그들 중에서는 A1B3형이 8건(5.1%)으로 가장 높은 빈도를 보였다. B306, cis-AB01,Ael02, Aw14 그리고 B305 대립유전자를 가진 5발단자의 가계조사에서 동반되는 ABO 대립유전자에 따라 가족들이 다른 표현형을 보임을 보여주었고, A102/B101/O04를 갖는 키메라는 전형적인 키메라 유전양상을 보여주었다. 결론: 저자들은 ABO 유전형 검사를 시행한 6년 간의 경험을 통해 ABO 불일치의 해결과 ABO 아형의 감별에 있어서 PCR-직접염기서열분석법이 유용함을 밝히는 바이다. 또한 다양한 ABO 아형의 표현 양상을 분석할 수 있는 가계조사가 함께 이뤄져야 할 것이다.

Distribution of HCV Genotypes in Chronic Korean HCV Patients

Lee, Kyung-Ok,Jeong, Su-Jin,Byun, Ji-Young,Shim, Ae-Sug,Seong, Hye-Soon,Kim, Kyung-Tae Korean Society for Clinical Laboratory Science 2007 대한임상검사과학회지(KJCLS) Vol.39 No.1

HCV is a single-stranded RNA virus and more than 1 million new cases are reported annually worldwide. The six major HCV genotypes and numerous subtypes vary in their geographic distribution. It is thought that genetic heterogeneity of HCV may account for some of the differences in disease outcome and response to treatment observed in HCV infected persons. In this study, we determined HCV genotypes among chronic Korean HCV patients and evaluated direct sequence PCR protocols developed. For the study, 232 chronic HCV patient sera were used. HCV RNA was extracted and two pairs of consensus PCR primers were selected in 5'UTR region for amplification of HCV RNA. Amplification products obtained from the HCV positive cases were subjected to automatic sequencing. Sequences were compared with those in GenBank by using the BLAST program. From this study, five HCV genotypes, 1b, 2a, 2b, 2c and 3a were found. HCV genotypes 4, 5 and 6 were not determined. HCV genotype 1b (53.9%, 125/232) and 2a (35.8%, 83/232) were most frequently found. This group was followed by 2b (3.9%, 9/232), 3a (3.4%, 8/232) and 2c (3.0%, 7/232). The data presented here suggest a complex distribution of HCV types and they were well correlated with other reports on Koreans and will be helpful for type-specific follow-up of Korean HCV patients. This study showed that 5'UTR direct sequence analysis is a sensitive and rapid method to identify HCV genotypes. HCV는 single stranded RNA 바이러스로서 감염 시에는 만성간염 및 간경화 간암으로 진행될 수 있는 가능성이 높다. HCV는 6종의 주된 genotype과 그에 따른 많은 종류의 subtype이 보고되고 있으며, 세계 각 지역별로 그 분포는 매우 다양하다. 여러 가지 HCV genotype 중에서 1b 형에 감염되었을 경우 간경화나 간암으로 진행할 가능성이 높으며 치료효과도 떨어진다는 보고가 있어, 최근 HCV 환자의 치료에 있어서 HCV 바이러스 정량검사와 함께 HCV genotyping 검사의 임상적 활용이 높아지고 있다. 본 연구에서는 PCR-direct sequencing을 이용한 HCV genotyping 검사방법을 이용하여, 한국인 만성 HCV 간염환자에서 HCV genotype의 분포를 조사하였다. 검체로는 232명의 한국인 만성간염환자의 혈청을 사용하였으며, HCV 5'UTR 영역에서 선택한 2쌍의 primer로 nested PCR을 실시하였다. 증폭된 PCR산물 (215 bps)은 2% agrose gel로 전기영동을 하고 sequencing을 실시한 후 GeneBank의 BLAST 프로그램을 사용하여 HCV genotype을 분석하였다. HCV genotyping을 실시한 232명에서 5종류의 genotype, HCV 1b, 2a, 2b, 2c, 3a, 이 발견되었으며, HCV genotype 4, 5, 6 은 검출되지 않았다. 발견된 HCV genotype 중에서 HCV 1b의 검출률이 53.9%로 가장 높았고, 다음은 HCV 2a가 35.8%로 높게 나타나, 위 두 가지 HCV genotype을 합하면 거의 90%였다. 다음으로 HCV genotype 2b가 3.9%, 3a가 3.4% 그리고 2c가 3.0%의 순서로 검출되었다. 본 결과는 한국인 만성 HCV간염 환자의 치료 및 예후관리에 참고가 될 것으로 사료된다. 또한 PCR-direct sequencing을 이용한 HCV genotyping 검사는 간편하고 분명하게 결과를 판독할 수 있어 임상실험실에서 유용하게 사용될 수 있을 것으로 판단된다.

Distribution of HCV Genotypes in Chronic Korean HCV Patients

( Kyung Ok Lee ),( Su Jin Jeong ),( Ji Young Byun ),( Ae Sug Shim ),( Hye Soon Seong ),( Kyung Tae Kim ) 대한임상검사과학회 2007 대한임상검사과학회지(KJCLS) Vol.39 No.1

HCV는 single stranded RNA 바이러스로서 감염 시에는 만성간염 및 간경화 간암으로 진행될 수 있는 가능성이 높다. HCV는 6종의 주된 genotype과 그에 따른 많은 종류의 subtype이 보고되고 있으며, 세계 각 지역별로 그 분포는 매우 다양하다. 여러 가지 HCV genotype 중에서 1b 형에 감염되었을 경우 간경화나 간암으로 진행할 가능성이 높으며 치료효과도 떨어진다는 보고가 있어, 최근 HCV 환자의 치료에 있어서 HCV 바이러스 정량검사와 함께 HCV genotyping 검사의 임상적 활용이 높아지고 있다. 본 연구에서는 PCR-direct sequencing을 이용한 HCV genotyping 검사방법을 이용하여, 한국인 만성 HCV 간염환자에서 HCV genotype의 분포를 조사하였다. 검체로는 232명의 한국인 만성간염환자의 혈청을 사용하였으며, HCV 5``UTR 영역에서 선택한 2쌍의 primer로 nested PCR을 실시하였다. 증폭된 PCR산물 (215 bps)은 2% agrose gel로 전기영동을 하고 sequencing을 실시한 후 GeneBank의 BLAST 프로그램을 사용하여 HCV genotype을 분석하였다. HCV genotyping을 실시한 232명에서 5종류의 genotype, HCV 1b, 2a, 2b, 2c, 3a, 이 발견되었으며, HCV genotype 4, 5, 6 은 검출되지 않았다. 발견된 HCV genotype 중에서 HCV 1b의 검출률이 53.9%로 가장 높았고, 다음은 HCV 2a가 35.8%로 높게 나타나, 위 두 가지 HCV genotype을 합하면 거의 90%였다. 다음으로 HCV genotype 2b가 3.9%, 3a가 3.4% 그리고 2c가 3.0%의 순서로 검출되었다. 본 결과는 한국인 만성 HCV간염 환자의 치료 및 예후관리에 참고가 될 것으로 사료된다. 또한 PCR-direct sequencing을 이용한 HCV genotyping 검사는 간편하고 분명하게 결과를 판독할 수 있어 임상실험실에서 유용하게 사용될 수 있을 것으로 판단된다. HCV is a single-stranded RNA virus and more than 1 million new cases are reported annually worldwide. The six major HCV genotypes and numerous subtypes vary in their geographic distribution. It is thought that genetic heterogeneity of HCV may account for some of the differences in disease outcome and response to treatment observed in HCV infected persons. In this study, we determined HCV genotypes among chronic Korean HCV patients and evaluated direct sequence PCR protocols developed. For the study, 232 chronic HCV patient sera were used. HCV RNA was extracted and two pairs of consensus PCR primers were selected in 5’UTR region for amplification of HCV RNA. Amplification products obtained from the HCV positive cases were subjected to automatic sequencing. Sequences were compared with those in GenBank by using the BLAST program. From this study, five HCV genotypes, 1b, 2a, 2b, 2c and 3a were found. HCV genotypes 4, 5 and 6 were not determined. HCV genotype 1b (53.9%, 125/232) and 2a (35.8%, 83/232) were most frequently found. This group was followed by 2b (3.9%, 9/232), 3a (3.4%, 8/232) and 2c (3.0%, 7/232). The data presented here suggest a complex distribution of HCV types and they were well correlated with other reports on Koreans and will be helpful for type-specific follow-up of Korean HCV patients. This study showed that 5’UTR direct sequence analysis is a sensitive and rapid method to identify HCV genotypes.

Detection of Lamivudine-Resistant Mutations of HBV DNA Polymerase Gene Using PCR-Direct Sequencing

( Kyung Ok Lee ),( Hye Jung Lee ),( Ji Young Byun ),( Sung Yeun Lee ),( Jeong Sook Kim ),( Na Young Jung ),( Soo Jin Chung ),( Hye Soon Seong ),( Kyung Tae Kim ) 대한임상검사과학회 2006 대한임상검사과학회지(KJCLS) Vol.38 No.3

Treatment of hepatitis B virus (HBV) with lamivudine is effective in suppressing virus replication and results in reduced inflammatory activity. However the most troublesome problem of lamivudine treatment is the emergence of lamivudine-resistant strains with amino acid substitution in the YMDD motif of DNA polymerase gene during the treatment. The aim of this study was to determine the mutation of YMDD motif (codon 552) and codon 528 in chronic HBV patients with lamivudine therapy using PCR-direct sequencing and to investigate the relationship between lamivudine mediated HBV mutation and HBeAg. HBV DNA was extracted from serum samples of HBV patients and amplified by nested PCR with two sets of primer pairs selected in HBV DNA polymerase gene. Amplified PCR product was analyzed by 2% agarose gel electrophoresis and direct sequencing. HBV mutation was detected in 124 out of 207 samples (60%). Single mutation was 50.8% for M552I, 43.5% for M552V, 5.7% for M552I/V and the L528M mutation was 67.0%. Double mutation was 43.6% for M552V/L528M, 33.1% for M552I/L528(wild type), 17.7% for M552I/L528M and 5.6% for M552I/V/L528M. Serine mutation at YMDD motif (M552S) was not found and the L528M mutation frequently accompanied M552V type. In this study, the typical difference of frequencies for HBV mutation depending on HBeAg was not found. Moreover, the PCR-direct sequencing method used in this study might be a powerful tool for the mutation study in clinical reference laboratories with high volume.

직접염기서열분석법을 이용한 한국인 Lewis 혈액형군의 유전자분석

송서영,안성수,유숙원,김장수,서인범 대한혈액학회 2008 Blood Research Vol.43 No.1

Background: The FUT2 and FUT3 genes determine the Lewis phenotype of red blood cells (RBCs). Recently, the Lewis genes, the secretor genes, and several mutations that cause Lewis negative and nonsecretor phenotypes have been identified. The purpose of this study was to analyze the gene frequency of FUT2 and FUT3 in a Korean population by comparing the use of the direct sequencing method to the use of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for mutation detection in the FUT2 and FUT3 genes. Methods: RBCs and peripheral blood leukocytes were obtained from 225 apparently healthy volunteers to determine the phenotype and genotype of the FUT2 and FUT3 genes. Lewis phenotypes were determined on K3EDTA-stablized fresh blood samples using the column agglutination method. Lewis blood group genotyping was performed by use of the direct sequencing method. For the detection of T59G, C357T, and A385T mutations, the PCR-RFLP method was performed. Results: The frequencies of the Lewis blood group phenotype were 12.4% for Le(a+b−), 70.7% for Le(a−b+), 11.1% for Le(a−b−) and 5.8% for Le(a+b+), respectively. Direct Sequencing of the FUT2 gene identified 92.2% C357T, 56.9% A385T, 0.4% G244A mutations and 1.8% del396. Direct Sequencing of the FUT3 gene identified 46.9% T59G, 30.4% G508A, 1.1% T202C, 1.1% C314T, 0.7% A1029G, 3.0% T1067A and 13.3% G1242A mutations. The PCR-RFLP method results were discordant in nine cases (1 case for C357T, 4 cases for A385T and 2 cases for T59G) as compared to the direct sequencing method results. Conclusion: We have determined the frequencies of FUT2 and FUT3 gene mutations in a Korean population. The use of the direct sequencing method was more accurate than the use of the PCR-RFLP method for the determination of the genotype of the FUT2 and FUT3 genes.

PCR 증폭과 직접 염기서열분석으로 진단한 Mycobacterium marinum에 의한 피부감염증

김진용 ( Jin Yong Kim ),서수현 ( Soo Hyun Seo ),황은정 ( Sun Jung Hwang ),최미라 ( Mi Ra Choi ),박성섭 ( Sung Suo Park ),섬문우 ( Moon Woo Seong ),조광현 ( Kwang Hyun Cho ) 대한피부과학회 2013 대한피부과학회지 Vol.51 No.9

Mycobacterium marinum is an atypical mycobacterium (ATM) and is an uncommon cause of skin and soft tissue infections associated with contact with contaminated water. Diagnosis is often delayed when only a conventional identification method is used. PCR amplification and direct sequencing is recently available method for rapid identification of ATM. We report a case of M. marinum infection identified by PCR and sequencing. A 56-year-old female was referred for multiple erythematous nodules on both forearms which appeared two months ago. Skin biopsy showed suppurative granulomatous inflammation, and AFB culture showed nontuberculous Mycobacteria. PCR and sequencing were performed, and the obtained sequences were compared to the database using BLAST. The sequences of 16S rRNA and rpoB could not differentiate between M. marinum and M. ulcerans, showing 100%homology to both. Identification was possible using the sequences of the tuf and hsp65 genes, showing both 100%homology to M. marinum, while 99.8%, 99.7% to M. ulcerans. The patient was treated with clarithromycin, rifampicin,and ethambutol for 6 months.

Retrieving Authentic DNA Barcoding Sequence from Old Insect Specimens

Taeman Han,Tae Hwa Kang,Oh Chang Kwon,Young Bo Lee,Mi Ae Kim,Hae Chul Park 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.10

In DNA barcoding, the DNA degradation of old museum specimens has been limited full-length (658bp) sequencing. The challenges associated with the retrieval and authentication of degraded DNA extracts from fossil and old museum specimens were principally limited to analyze the relatively short sequences (<300 bp). Furthermore, almost protocols in other to analyzed the degraded DNA contained the cloning process after PCR causing the time-consuming and the rising costs. To overcome these problematic circumstances, we tried a modified method to analyze full-length of DNA barcoding region in 30~60 year-old butterfly specimens (225 samples in 28 species), using direct sequencing after PCR with species-specific overlapping primer sets per each species. As a result, all of 28 species have been successfully analyzed, although 178 samples (79%) are completely generated barcoding sequences ranged from 640 to 658 bp and 47 samples (21%) are partially sequenced ranged from 100 to 500 bp. Thus, the result showed that the direct PCR sequencing using the overlapping primer sets per species appears to have great potential efficiency for analysis of degraded DNA without incorrect sequences.