• BMB Reports
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• 제46권5호
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• pp.270-275
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• 2013

It is essential to analyze rare mutations in many fields of biomedical research. However, the detection of rare mutations is usually failed due to the interference of predominant wild-type DNA surrounded. Herein we describe a sensitive and facile method of detecting rare point mutation on the basis of allele-specific amplification in emulsion PCR. The identification and selective amplification of rare mutation are accomplished in one-pot reaction. The allele-specific primers coupled on magnetic beads allow the exclusive amplification and enrichment of the mutant amplicons. The productive beads bearing mutant amplicons are subsequently stained with the fluorescent dyes. Thus, the rare point mutations with a percentage as low as 0.1%, can be detected by fluorescent analysis. The relative percentages of mutation among different samples can be roughly accessed by counting the fraction of fluorescent positive beads through flow cytometry.

• 한국동물생명공학회지
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• 제38권4호
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• pp.209-214
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• 2023

Background: Single nucleotide polymorphisms (SNPs) are widely used genetic markers with applications in human disease diagnostics, animal breeding, and evolutionary studies, but existing genotyping methods can be labor-intensive and costly. The aim of this study is to develop a simple and rapid method for identification of a single nucleotide change. Methods: A modified Polymerase Chain Reaction Amplification of Multiple Specific Alleles (PAMSA) and high resolution melt (HRM) analysis was performed to discriminate a bovine polymorphism in the NCAPG gene (rs109570900, 1326T > G). Results: The inclusion of tails in the primers enabled allele discrimination based on PCR product lengths, detected through agarose gel electrophoresis, successfully determining various genotypes, albeit with some time and labor intensity due to the use of relatively costly high-resolution agarose gels. Additionally, high-resolution melt (HRM) analysis with tailed primers effectively distinguished the GG genotype from the TT genotype in bovine muscle cell lines, offering a reliable way to distinguish SNP polymorphisms without the need for time-consuming AS-PCR. Conclusions: Our experiments demonstrated the importance of incorporating unique mismatched bases in the allele-specific primers to prevent cross-amplification by fragmented primers. This efficient and cost-effective method, as presented here, enables genotyping laboratories to analyze SNPs using standard real-time PCR.

• BMB Reports
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• 제32권6호
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• pp.529-534
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• 1999

Asymmetric PCR and single-strand conformation polymorphism (SSCP) methods were combined to analyze human leukocyte antigen (HLA)-DRB allele polymorphism. Asymmetric PCR amplification was applied to generate single-stranded DNA (ssDNA) using the nonradioactive oligonucleotide primers desinged for the polymorphic exon 2 region. The conformational differences of ssDNAs, depending on the allele type, were analyzed by nondenaturing polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The ssDNAs were clearly separated from double-stranded DNA without interference and obviously migrated depending on their allele type. This method was applied to the genomic DNA either from homozygous or from heterozygous cell lines containing the DR4 allele as template DNA using DR4-specific primers, and satisfying results were obtained. Compared to the standard PCR-SSCP method, this asymmetric PCR-SSCP method has advantages of increased speed, reproducibility, and convenience. Along with PCR-SSP or sequence-based typing, this method will be useful in routine typing of HLA-DRB allele.

• 약학회지
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• 제37권5호
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• pp.504-510
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• 1993

Human insulin gene is consisted of the polymorphic region with the repeating units, the regulatory sequence, the structural gene including the intervening sequence, and 3'-flanking region. The polymerase chain reaction, which amplifies the target DNA between two specific primers, has been performed for the amplification of human insulin gene and simple one-step cloning of it into Escherichia coli. Out of 1727 nuceotides compared, only 4 sites were variable: 5'-regulatory region(G2101$\rightarrow$AGG); IVS I(T2401$\rightarrow$A); Exon II(C2411 deletion); IVS II(A2740 dejection). The variations at the G2101 and T2401 were the same as those found in one American allele. The other two variations were observed only in the specific Korean allele. And, the enzyme digestion patterns among normal, insulin dependent diabetes mellitus, and non-insulin dependent diabetes mellitus were the same. On the other hand, PCR method showed the possibility of the quickaccess for the polymorphic region in terms of the restriction fragment length of polymorphism.

• BMB Reports
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• 제28권4호
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• pp.359-362
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• 1995

The highly polymorphic variable number of tandem repeat (VNTR) loci in the human genome are informative markers for the genetic characterization of individuals in the paternity test and forensic science as well as for the study of human disease. In this study, VNTR loci D1S80 and D2S123 have been amplified by PCR and the amplified length polymorphic alleles were detected with a discontinuous vertical PAGE system and silver staining. For explicit DNA typing, PCR optimization, in which amplification efficiencies are similar over a wide range of allele sizes, non-specific amplifications are minimal, and new longer alleles have high amplification efficiency, has been performed by changing the PCR reaction buffer composition and thermal cycling conditions. It turned out that adding an appropriate amount of Tween 20 and NP40 to the PCR reaction buffer and raising the annealing temperature to $68^{\circ}C$ in thermal cycling made it possible for optimal VNTR loci amplification. A modified PAGE system for VNTR separation was established. Under these conditions, new longer alleles in the 01580 locus were discovered and 025123 pattern changes in colorectal tumors were observed. These technical tips are valuable for detecting various amplified fragment length polymorphisms.

• Clinical and Experimental Reproductive Medicine
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• 제40권4호
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• pp.163-168
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• 2013

Objective: Preimplantation genetic diagnosis (PGD) is an assisted reproductive technique for couples carrying genetic risks. Charcot-Marie-Tooth (CMT) disease is the most common hereditary neuropathy, with a prevalence rate of 1/2,500. In this study, we report on our experience with PGD cycles performed for CMT types 1A and 2F. Methods: Before clinical PGD, we assessed the amplification rate and allele drop-out (ADO) rate of multiplex fluorescent polymerase chain reaction (PCR) followed by fragment analysis or sequencing using single lymphocytes. We performed six cycles of PGD for CMT1A and one cycle for CMT2F. Results: Two duplex and two triplex protocols were developed according to the available markers for each CMT1A couple. Depending on the PCR protocols, the amplification rates and ADO rates ranged from 90.0% to 98.3% and 0.0% to 11.1%, respectively. For CMT2F, the amplification rates and ADO rates were 93.3% and 4.8%, respectively. In case of CMT1A, 60 out of 63 embryos (95.2%) were diagnosed and 13 out of 21 unaffected embryos were transferred in five cycles. Two pregnancies were achieved and three babies were delivered without any complications. In the case of CMT2F, a total of eight embryos were analyzed and diagnosed. Seven embryos were diagnosed as unaffected and four embryos were transferred, resulting in a twin pregnancy. Two healthy babies were delivered. Conclusion: This is the first report of successful pregnancy and delivery after specific PGD for CMT disease in Korea. Our PGD procedure could provide healthy babies to couples with a high risk of transmitting genetic diseases.

• Genomics & Informatics
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• 제20권2호
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• pp.18.1-18.7
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• 2022

According to long-term projections, by 2030, the world's population is predicted to reach 7.5 billion individuals, and there will be roughly 27 million new cancer cases diagnosed. The global burden of breast cancer (BC) is expected to rise. According to the Ministry of Health-Iraqi Cancer Registry, cancer is the second largest cause of death after cardiovascular disease. This study investigated the interleukin-18 (IL18) single-nucleotide polymorphisms (SNPs) -607C/A rs1946518 and -137G/C rs187238 using the sequence-specific amplification-polymerase chain reaction approach. Regarding the position -607C/A, there was a highly significant difference between the observed and expected frequencies in patients and controls (χ² = 3.16 and χ² = 16.5), respectively. The AA and CA genotypes were associated with significantly increased BC risk (odds ratio [OR], 3.68; p = 0.004 and OR, 2.83; p = 0.04, respectively). Women with the A allele had a 5.03-fold increased susceptibility to BC. The C allele may be a protective allele against BC (OR, 0.19). Although position -137G/C showed no significant differences in the CC genotype distribution (p = 0.18), the frequency of the CC genotype was significantly higher in patients than in controls. In contrast, patients had a significantly higher frequency of GC genotypes than controls (p = 0.04), which was associated with an increased risk of developing BC (OR, 2.63). The G allele frequency was significantly lower in patients than in controls (55.0% vs. 76.2%, respectively). This SNP may be considered a common genotype in the Iraqi population, with the wild-type G allele having a protective function (OR, 0.19) and the mutant C allele having an environmental effect (OR, 2.63).

• Journal of Genetic Medicine
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• 제5권1호
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• pp.41-46
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• 2008

목 적 : 취약 X 증후군(fragile X syndrome)은 FMR1 유전자의 5' 비해독부위에 있는 CGG 3염기 반복의 확장에 의해 발생되는 유전성 질환이다. 방 법 : 본 연구에서는 임상 소견과 핵형분석에서 취약 X 증후군으로 진단 받은 여아 환자와 그 부모를 대상으로 Abbott Molecular Fragile X PCR Kit를 이용하여 CGG 3염기 영역을 PCR로 증폭하여 normal, premutation, full mutation의 CGG 반복의 유형을 확인하였으며, premutation과 normal allele의 경우에는 정확한 CGG 반복수를 분석하였다. 결 과 : 환자는 30회와 >200회의 CGG 3염기가 반복된 FMR1 대립유전자를 갖고 있는 것으로 확인되어 취약 X 증후군으로 진단되었다. 또한 환자의 어머니에서 30과 98회의 반복 allele을 확인함으로써, 이 환자의 full mutation allele은 모계의 premutation allele로부터 유래한 것임을 알 수 있었다. 결 론 : Abbott Molecular Fragile X PCR Kit를 사용한 진단방법은, 취약 X 증후군환자의 경우에서 통상적으로 시행되고 있는 PCR, MS-PCR, Southern blotting을 병행하는 방법에 비해 신속하고 정확한 분자유전학적 진단이 가능한 유용한 방법이라 생각된다.

• Asian-Australasian Journal of Animal Sciences
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• 제19권5호
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• pp.622-626
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• 2006

Molecular characterization of Hallikar, the native cattle breed of Karnataka, was undertaken using 19 cattle specific, highly polymorphic microsatellite markers recommended by FAO. The genomic DNA was subjected to PCR amplification and alleles were resolved through six per cent denaturing PAGE with a 10 bp DNA ladder followed by silver staining. Genotyping of animals was done based on allele size. The number of alleles ranged from three to nine with allele sizes ranging from 102 bp to 294 bp. These alleles were distributed in the frequency range between 0.0306 and 0.8673 in the population. The mean observed number of alleles was $6.368{\pm}1.4225$. The mean observed and expected heterozygosities were $0.7515{\pm}0.1734$ and $0.7850{\pm}0.1381$, respectively. The high heterozygosity observed implies presence of higher genetic variability within Hallikar breed. The PIC (Polymorphism Information Content) values ranged from 0.2322 (ETH152) to 0.8654 (ETH225). The percentage of polymorphic loci obtained was 100 as all the 19 microsatellite markers were found to be polymorphic. Except for ETH152, all the other loci had high PIC values, indicating that these markers are highly informative for characterization of Hallikar breed. The population was tested for Hardy-Weinberg equilibrium at 19 microsatellite loci, and at 74 per cent of the loci the population was found to be in disequilibrium.

• Asian Pacific Journal of Cancer Prevention
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• 제15권4호
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• pp.1575-1579
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• 2014

Background: There is a close relationship between Helicobacter pylori (H pylori)-specific factors and different gastroduodenal diseases. The present study aimed to investigate the prevalence of vacA d1, d2 genotypes in the H pylori isolates from patients with gastric adenocarcinoma, peptic ulcer disease (PUD) and gastritis in East Azerbaijan region, where the incidence of gastric cancer (GC) is high. Strains isolated from this area are likely to be of European ancestry. Materials and Methods: In this study, genotyping of the vacA d region of 115 isolates obtained from patients with different gastrodoudenal diseases was accomplished by PCR methods. In addition to PCR amplification of H pylori 16S rDNA, rapid urease tests or histological examination were used to confirm the presence of H pylori in biopsy specimens. Data were collected and analyzed using SPSS version 19. Results: Of the total of 83 H pylori isolates, 36 (43.4%) contained the d1 allele and 47 (56.6%) were subtype d2. The results of the multiple linear/logistic regression analysis showed high correlation between allele d1 and gastric adenocarcinoma or PUD. Conclusions: This study suggests that the H pylori vacA d1 genotype helps predict risk for gastric adenocarcinoma and PUD in East Azerbaijan, Iran.