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ObjectiveTo establish a polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） method for rapid distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex， so as to avoid the influence of genetic confusion on drug safety. MethodThe DSS-tagged sequences of Periplocae Cortex were obtained from the Chloroplast Genome Information Resource （CGIR） and analyzed to find the enzymatic cleavage sites that were different from those of Acanthopanacis Cortex and Lycii Cortex. The specific enzymatic cleavage site， Cla I， of Periplocae Cortex was selected， on the basis of which the primers for PCR-RFLP were designed. Furthermore， the factors such as annealing temperature， number of cycles， Taq enzyme， PCR instruments， and enzymatic treatment time that may influence PCR-RFLP were studied. The established PCR-RFLP method was applied to the identification of Periplocae Cortex， Acanthopanacis Cortex， and Lycii Cortex samples produced in different regions. ResultThe PCR-RFLP at the annealing temperature of 59 ℃ and with 40 cycles showed clear bands of the samples. When the enzyme digestion time was 30 min. The reaction produced the target bands at about 140 bp and 290 bp for both Periplocae Cortex and its original plant and only a band at about 430 bp for Acanthopanacis Cortex， Lycii Cortex， and their original plants. The method can accurately distinguish Periplocae Cortex from its confounders Acanthopanacis Cortex and Lycii Cortex. ConclusionThe PCR-RFLP method for distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex was established. It has high stability， sensitivity， and applicability， providing a reference for the quality control of Periplocae Cortex， Acanthopanacis Cortex， and Lycii Cortex.
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ObjectiveTo establish a rapid method for evaluating the heterozygosity of Murraya paniculata germplasm materials and provide as a foundation for developing germplasm breeding and innovation measures for M. paniculata. MethodSingle nucleotide polymorphisms （SNPs） were screened from the genome resequencing data of 65 plants of M. paniculata. A self-written script was used to transform 20 SNPs into restriction fragment length polymorphism （RFLP） markers. Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） was employed to detect the 20 RFLP markers in 12 M. paniculata germplasm accessions， and the heterozygosity of M. paniculata germplasm accessions was calculated based on the number of enzyme-cutting bands at the 20 RFLP marker sites. Plink was used to calculate the whole genome heterozygosity of 12 M. paniculata germplasm accessions， and the results obtained with different methods were compared. ResultThere was no significant difference in the heterozygosity calculated by the PCR-RFLP method and the genome resequencing method. The PCR-RFLP and genome resequencing methods identified 8 and 9 germplasm accessions， respectively， with a heterozygosity level less than 30%. Seven germplasm accessions with heterozygosity less than 30.00% were calculated by both methods. ConclusionThe PCR-RFLP method established in this study for evaluating the heterozygosity of M. paniculata germplasm demonstrates the precision of 87.5% and the accuracy of 77.8%. This method serves as a reference for developing heterozygosity evaluation methods in other medicinal plant germplasm resources.
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ObjectiveIn recent years， with the sharp decline of wild resources in Arisaematis Rhizoma and Pinelliae Rhizoma and the immaturity of medicinal cultivation technology， their adulterants have appeared frequently in the market， and the main identifying characteristics have mostly disappeared in the circulation of medicinal materials. Therefore， there is an urgent need to establish a molecular identification method that can quickly and effectively identify the specificity of Arisaematis Rhizoma and Pinelliae Rhizoma. MethodAfter comparison of the rbcL sequences of Arisaematis Rhizoma，Pinelliae Rhizoma， and their adulterants， the specific enzyme cleavage sites Hae Ⅲ and Dra Ⅰ of Arisaematis Rhizoma and Pinelliae Rhizoma， respectively， were selected and identified by polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP）. The main system conditions of PCR-RFLP reaction were established and optimized， and their durability and the ability to detect genuine， adulterants， and mixed counterfeits were investigated. ResultThe PCR-RFLP identification method of Arisaematis Rhizoma and Pinelliae Rhizoma was established. After specific primer amplification， Arisaematis Rhizoma and Pinelliae Rhizoma could be digested by Hae Ⅲ and Dra Ⅰ-restricted endonucleases respectively， at annealing temperature of 54 ℃， the number of cycles of 35， and the amount of DNA template of 3-30 ng， producing two fragments or small cut fragments with a single band between 100-250 bp， whereas the mixed counterfeits were not cleaved and both showed a band at 250 bp. The method is highly accurate in identifying adulterants and mixed counterfeits of Arisaematis Rhizoma or Pinelliae Rhizoma. ConclusionThe PCR-RFLP method developed in this study allows for the rapid identification of Arisaematis Rhizoma and Pinelliae Rhizoma.
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Background and Aim of Study: The role of E-cadherin (CDH1) gene-160 C>A (rs16260) promoter polymorphism in colorectal cancer (CRC) still remains inconclusive. The aim of this study is to investigate the associations between the CDH1-160 C>A polymorphism with the susceptibility and clinicopathological development of CRC in the Turkish patients. To our knowledge, this is the first report examining the role of CDH1 polymorphism in Turkish CRC patients. Materials and Methods: A total of 92 colorectal carcinoma cases (including 62 colon and 30 rectal cancer patients) and the corresponding adjacent normal tissues as controls were studied. The polymorphism was genotyped using polymerase chain reaction-restriction fragment length polymorphism analysis. Clinicopathological features including patient's age, gender, tumor stage, and tumor location (colon/rectum) were compared statistically with the polymorphism status. Results: There was no significant difference in both genotype and allele frequencies of the CDH1 polymorphism between colorectal tumor cases and normal samples (P = 0.472 and 0.508, respectively). Furthermore, no significant associations were observed between the CDH1 polymorphism status and age, gender, tumor stage, and tumor location of the colorectal tumor cases (all P > 0.05). Conclusions: These results indicate that CDH1-160 C>A polymorphism does not contribute to the genetic susceptibility of CRC and the polymorphism may not be a direct effect on the progression of the disease in Turkish CRC patients.
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Objective To preliminary study the distribution of CYP2D6 polymorphism in Hubei popula-tion ,with the intention of solid base for further applied research .Methods Venous blood was collected from 137 volunteers ,analysed CYP2D6 * 10 Gene Polymorphism by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method .genotypes can be distinguished by agarose gel electro-phoresis and gene sequencing .Hardy-Weinberg equilibrium law could be used to detect whether the genotype distribution was balanced ,and chi-square test was used for verification ,and the results were compared with the previous literature .Results Among the 137 samples ,wild type (CC) was 35 ,the frequency was 25 .5% .Het-erozygote (CT) was 52 ,the frequency was 38 .0% .And mutant (TT) was 50 ,the frequency was 36 .5% .Also , Callele frequency was 44 .5% while the Tallele was 55 .5% .There was no statistical difference compared with previous studies (P＞0 .05) .Conclusion Since there is a high mutation frequency of CYP2D6*10 in Hubei population ,it is very necessary to carry out genotyping to guide clinical medication correctly .
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@#Objective To identify the genotype of Toxoplasma gondii isolated strains from a congenital teras（KS strain）and an HIV⁃Toxoplasma co⁃infected patient in Jiangsu Province. Methods T. gondii DNA of tachyzoites of a isolate from a congenital teras（KS strain）and blood DNA of an HIV⁃Toxoplasma co⁃infected patient in Jiangsu Province were extracted，and 11 loci were identified for the genotype by polymerase chain reaction restriction fragment length polymorphism（PCR⁃RFLP）. Results The complete bands were obtained from the congenital teras（KS strain）and HIV⁃Toxoplasma co⁃infected patient in Jiangsu Province，and identified as T. gondii gene type I. Conclusion T. gondii gene type I may be the dominant genotype strain of T. gondii among the women who have the abnormal pregnant outcomes and HIV⁃Toxoplasma co⁃infected patients in Jiangsu Province.
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Objective To investigate the correlation between KLF14 gene rs4731702 locus polymorphism and gestational diabe-tes mellitus (GDM) and relation between its different genotypes with BMI and insulin resistance.Methods This study adopted the case-control method.One hundred pregnant women of GDM (GDM group) and one hundred healthy pregnant women (normal con-trol group) were randomly selected as the research subjects and performed the physical examination ,biochemical indicators detec-tion.HOMA-IR and HOMA-β were calculated.The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was adopted.The genotyping of KLF14 gene rs4731702 locus in the two groups was performed.The genotypes and allele gene frequency were compared between the two groups and the GDM association analysis was conducted.Results The C alleles fre-quency and CC genotype frequency of KLF14 gene rs4731702 locus in the GDM group was significantly higher than that in the con-trol group ,the difference was statistically significant (P< 0.05).The patients with genotype CC in the GDM group had higher BMI ,FPG ,TG ,HbA1c and HOMA-IR as compared with those carrying genotype CT and TT ,the difference was statistically signif-icant(P<0.05).Also they had lower FINS ,HDL and HOMA-βas compared with carriers of genotype CT and TT ,the difference was statistically significant(P<0.05).The multivariate analysis showed that the genotype CC of KLF14 gene rs4731702 locus was closely related with GDM (P=0.005 ,RR=25.128).Conclusion The genotype CC of KLF14 gene rs4731702 locus plays a role in the pathogenesis process of GDM ,may be susceptibility genes for GDM ,which is also related to the abnormal lipid metabolism ,islet dysfunction and obesity.The polymorphism study of KLF14 gene rs4731702 locus helps to reveal the relation between lipid metabo-lism abnormality and insulin resistance with GDM onset.
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Introduction@#Base excision repair (BER) is mainly responsible for the correction of small base changes of DNA damage. BER pathway involved many enzymes including OGG1 and XRCC1. The defective DNA repair is associated with an increased risk of various cancers including hematologic malignancies-leukemia and myelodysplastic syndrome (MDS). However, it is deniably these polymorphisms alter the susceptibility and clinical outcome of MDS patients.@*The aim@#This study was to evaluate the impact of polymorphisms in gene encoding one protein of BER system: XRCC1 Arg399Gln in MDS and healthy population.@*Methods@#In this study, we recruited 60 health control group [median 47.9 years, 9 MDS subjects [median 56.6 years] were included in this study. Genotyping was carried out by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Allele and genotype frequencies were calculated by direct counting.@*Result@#The frequencies of genotypes of XRCC1 Arg399Gln were as follows: Arg /Arg 1 (11%), Arg/Gln 6 (66%), Gln/Gln 2 (22%) in MDS and Arg /Arg 18.4%, Arg/Gln40%, Gln/Gln41.6% in health control for XRCC1 Arg399Gln. The result revealed that genotypes Arg399Gln increased the risk of MDS@*In conclusion@#this study is the first to analyze XRCC1 SNPs and their associated risk of MDS in Mongolian samples. To fully understand the role of DNA damage and DNA repair in the MDS, prospective studies are needed and other genes (OGG1 Ser326Cys, MUTYH Gln324His, APE Asp148Glu) of base excision repair pathway should be analyzed.
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Background and purpose:miR-196a2 functions as an oncogene during tumor initiation and pro-gression. The up-regulation promotes tumor cell proliferation, invasion and metastasis. Therefore, it is promising to be an important tumor biomarker. The aim of this study was to investigate whether rs11614913, a gene polymorphic site ofmiR-196a2, is associated with the risk of leukemia.Methods:A case-control analysis was employed. Bone marrow or periph-eral blood was collected from 210 leukemia patients diagnosed from Jan. 2009 to Jul. 2015 in Yantaishan Hospital (case group) as well as 250 healthy people who were physically examined during the same period (control group). Polymerase chain reaction-restriction fragment length polymorphism (PCR-PFLP) was used to detect the genotype of rs11614913. Application test was used to compare the difference in the frequency of each genotype between case group and control group. The odds ratio (OR) of SNP allelic genes was calculated using logistic regression analysis and 95%CI represented the risk of leukemia for each genotype.Results:The distribution differences in the frequency of T/T, C/C, C/T genotype of miR-196a2 rs11614913 between case group and control group were statistically significant (P<0.05). The risk of leukemia for individuals who carried mutant homozygous C/C was 2.661-fold higher than those carried wild-type homozygous T/T, and the difference was statistically significant (P<0.05).Conclusion:ThemiR-196a2 gene polymorphic site rs11614913 was associated with the risk of leukemia. Mutant homozygous C/C or C allelic gene carrying was probably a risk factor for leukemia.
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PURPOSE: The endothelial nitric oxide synthase (eNOS) G894T polymorphism has been reported to cause endothelial dysfunction and may have a role in the development of coronary artery disease (CAD). The aim of the present study was to investigate the association of eNOS G894T genetic polymorphism and plasma levels of nitric oxide (NO) with CAD risk in an Iranian population. MATERIALS AND METHODS: We studied 200 patients with angiographically documented CAD and 100 matched controls. Analysis of G894T genetic polymorphism of eNOS was performed by polymerase chain reaction-restriction fragment length polymorphism method. Plasma levels of NO were determined using Griess method. Biochemical analysis was conducted by routine colorimetric methods. RESULTS: Plasma levels of NO were significantly lower in CAD patients than control subjects (41.60±12.70 vs. 55.48±16.57, P=0.001). Also, the mean plasma levels of NO were significantly lower in T allele carriers of eNOS G894T polymorphism than G allele carriers (P0.05). CONCLUSION: Reduced plasma level of NO is associated with increased risk of CAD in our population. Moreover, eNOS G894T polymorphism is a significant risk factor for CAD development via reducing the plasma levels of NO. However, eNOS G894T polymorphism is not a contributing factor for the severity of CAD.
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Humans , Alleles , Coronary Artery Disease , Coronary Vessels , Gene Frequency , Genotype , Methods , Nitric Oxide Synthase Type III , Nitric Oxide , Plasma , Polymorphism, Genetic , Risk FactorsABSTRACT
BACKGROUND: Vitiligo is an autoimmune polygenic disorder characterized by loss of pigmentation due to melanocyte destruction. The PTPN22 gene +1858 C>T single nucleotide polymorphism (rs2476601) has been shown to be associated with various autoimmune disorders. OBJECTIVE: The aim of this study was to investigate whether the PTPN22 gene +1858 C>T single nucleotide polymorphism is associated with susceptibility to generalized vitiligo in a Turkish population. METHODS: One hundred and seven patients with generalized vitiligo, and one hundred and twelve gender-, age-, and ethnic-matched controls were enrolled in the study. Genotyping was done by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The PTPN22 +1858 C>T genotype and allele frequencies of the generalized vitiligo patients did not differ significantly from those of healthy controls. CONCLUSION: We found no association between the PTPN22 +1858 C>T gene polymorphism and vitiligo susceptibility in Turkish generalized-vitiligo patients.
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Humans , Gene Frequency , Genotype , Melanocytes , Pigmentation , Polymorphism, Single Nucleotide , VitiligoABSTRACT
Objectives: The aim of this investigation was to simultaneously detect and differentiate Mycoplasma genitalium and Ureaplasma urealyticum in female patients suffering from genital complications by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Materials and Methods : Genital swabs were taken from 210 patients. They were transported to the laboratory in phosphate-buffered saline. For PCR, samples were analysed with genus-specific MyUu-R and MyUu-F primers. This primer set, which was originally designed in our laboratory, amplified a 465 bp fragment (M. genitalium) and a 559 bp fragment (U. urealyticum). Samples containing a band of the expected sizes for the Mycoplasma strains were subjected to digestion with a restriction endonuclease enzyme of TaqI and Cac8I. Results: Of the 210 samples, a total of 100 (47.6%) samples were found to be positive for Mycoplasmas (seven M. genitalium isolates, 3.3%; and 89 U. urealyticum isolates, 42.4%), and coinfections with both species were detected in four samples (1.9%). The PCR-RFLP results showed that M. genitalium and U. urealyticum are different by enzyme patterns. Conclusion: PCR-RFLP offers a rapid and easily applicable protocol to simultaneous detection and differentiation of M. genitalium and U. urealyticum from clinical samples when specific primers and restriction enzymes are used.
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Adult , Aged , Bacteriological Techniques/methods , Diagnosis, Differential , Female , Genitalia, Female/microbiology , Humans , Middle Aged , Mycoplasma Infections/diagnosis , Mycoplasma Infections/microbiology , Mycoplasma genitalium/classification , Mycoplasma genitalium/genetics , Mycoplasma genitalium/isolation & purification , Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment Length , Pregnancy , Pregnancy Complications, Infectious/diagnosis , Pregnancy Complications, Infectious/microbiology , Time Factors , Ureaplasma Infections/diagnosis , Ureaplasma Infections/microbiology , Ureaplasma urealyticum/classification , Ureaplasma urealyticum/genetics , Ureaplasma urealyticum/isolation & purificationABSTRACT
Objective:To study the correlation of C(-938)A single nucleotide polymorphism (SNP) in the promoter of anti-apoptosis gene Bcl-2 with the clinical biological parameters of breast cancer patients in Hebei Province. Methods:Three genotypes(AA, AC, CC) of Bcl-2 C(-938)A from 113 samples of breast cancer patients were analyzed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, and the results were associated with clinical biological parameters. The distribution of genotype frequency was compared between different groups. Results:When stratified for axillary lymph node metastases, the frequency of AA genotype were 26.8%, 47.8% and 52.6% and the distribution of AC+CC genotypes were 73.2%, 52.2% and 47.4% in negative group, 1-3 metastasis group, and ≥4 metastasis group. The difference between the two groups was significant (χ~2=6.337, P=0.042). Compared with the AC+CC genotypes, the OR value of AA genotype in ≥4 metastasis group was 3.041 (95%CI=1.072-8.626). The frequency of AA genotype were 30.9% and 69.1% in gradeⅠ-Ⅱ group and grade Ⅲ group, and the frequency of AC+CC genotypes were 57.9% and 42.1%. The difference between the two groups was significant (χ~2=5.055; P=0.025). Compared with the AC+CC genotypes, the OR value of AA genotype in differentiated tumors(grade Ⅲ)was 3.082 (95%CI=1.122-8.465). Stratified for estrogen receptor (ER), progesterone receptor (PR) and C-erbB2, there was no difference between the distribution of AA genotype and AC+CC genotypes (χ~2=3.005, χ~2=1.504, χ~2=1.163, P>0.05). Conclusion:The AA genotype of Bcl-2 gene C(-938)A maybe correlated with high lymph node metastasis rate and poor differentiation.
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Purpose : Malassezia yeasts are globally distributed agents of pityriasis versicolor and are implicated in the pathogenesis of seborrhoeic and atopic dermatitis. The aim of this study is to identify the Malassezia species obtained from pityriasis versicolor patients, using morphological, biochemical, physiological as well as Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) methods. Materials and Methods: The identification of Malassezia species is performed according to microscopic features and physiological characteristics, including catalase reaction and Tween assimilation tests. The DNA is extracted from cultured Malassezia using the glass bead, phenol-chloroform method. The internal transcribed spacer 1(ITS1) region is amplified and there is restricted digestion of the PCR products with two enzymes Cfo I and Bst F5I. Results : The most commonly isolated species is M. globosa (47.6%). RFLP analysis of the PCR products of the ITS1 region is in complete agreement with those from the DNA sequences of the internal transcribed spacer (ITS) 1 region and the biochemical tests. Conclusion : Based on the findings of this study, it can be concluded that PCR-RFLP is a relatively simple and quick method, completely comparable to the routine methods used for Malassezia identification.
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Objective To create and evaluate the PCR restriction fragment length polymorphism (PCR-RFLP) based on hsp65 gene as a method for rapid identification of Mycobacteria to the species level. Methods hsp65 gene was amplified from the DNA of mycobacterial reference strains and the PCR products were subjected to digestion by two restriction endonucleases Hae Ⅲ and Bstp Ⅰ, then loaded onto a 4% MetaPhor agorose. The size of the restricted fragments of each species (strains) was determined according to the position of the fragments on the gel, by which the differential DNA fingerprint was confirmed. Results A total of 40 Mycobacterium species (strains) was analyzed, in which six reference strains of Mycobacterium tuberculosis Complex had two different electrophoresis patterns, and thirty-four reference species of non-tuberculosis Mycobacteria had unique pattern. Conclusion PCR-RFLP Based upon hsp65 gene can be used for identification of Mycobacterium species, and the method is more rapid and simple and easy-to-use for mycobacterial species identification.
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Leishmania (Viannia) peruviana was isolated from 1/75 Lutzomyia peruensis captured during May 2006 in an endemic cutaneous leishmaniasis region of the Peruvian Andes (Chaute, Huarochiri, Lima, Peru). Sand fly gut with promastigotes was inoculated into a hamster and the remaining body was fixed in ethanol. L. (Viannia) sp. was determined by polymerase chain reaction (PCR), and Leishmania species through molecular genotyping by PCR-restriction fragment length polymorphism analyses targeting the genes cpb and hsp70, resulting L. (V.) peruviana. The infected sand fly appeared 15 days after the rains finished, time expected and useful real time data for interventions when transmission is occurring.
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Animals , Cricetinae , Female , Male , DNA, Protozoan/analysis , Leishmania braziliensis/isolation & purification , Psychodidae/parasitology , Genotype , Leishmania braziliensis/genetics , Peru , Polymerase Chain Reaction , Polymorphism, Restriction Fragment LengthABSTRACT
In order to investigate the association of G+1688A (Ser563Asn) polymorphism of platelet endothelial cell adhesion molecule-1 (PECAM-1) gene with myocardial infarction (MI) in the Chi- nese Han population, the G+1688A polymorphism in PECAM-1 gene was detected by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) method among 502 subjects, including 218 patients with MI and 284 controls. The results showed that there was significant dif-ference in AA frequencies of genotype G+1688A polymorphism between case and control groups (39% vs 24%, P<0.001). A similar trend was observed on the allele frequencies (A/G: 62% vs 49%, P<0.001). Among the subjects with high serum total cholesterol level or high systolic blood pressure level, the variant AA genotype was associated with high risk of MI (adjusted OR, 2.13; 95% CI, 1.08-4.41 and adjusted OR, 2.53; 95%CI, 1.63-3.63). The single nucleotide polymorphism (SNP) at position +1688 in the exon 8 of PECAM-1 gene was associated with MI and the allele A might be a risk factor for MI in the Chinese Han population.
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0.05).Conclusion PAF-AH-Ala379Val gene mutation is unrelated to bronchial asthma in children.
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Objective:To investigate the gene polymorphism distribution of tumor necrosis factor-beta(TNF?) with SLE patient and different race in Chinese Han population of Jiangsu province.Methods:DNA samples were extraced from 168 EDTA-blood of unrelated healthy individuals and 66 SLE patients.TNF? alleles were typed using polymerase chain reaction-restrication fragment length polymorphism(PCR-RFLP). Results:The alleles frequencies of TNF? was higher significantly in Chinese Han population than in the White race;the gene frequencies of the TNF?*2 was higher significantly in SLE patients than in the normal controls(SLE patients 67.4%,normal controls 55.1%,P
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Apolipoprotein E (ape E) has three common alleles (ape epsilon 2, epsilon 3 and epsilon 4) that code for three major isoforms E2, E3 and E4. The isoforms differ from each other by a single amino acid substitutions at two positions and also differ in their binding affinity for the apo E receptors. Moreover, recently a strong association between the apo epsilon 4 allele and late-onset Alzheimer disease (AD) was demonstrated. In this study, were analyzed the apo E genotypes using the Hhal digestion of PCR amplified samples, and the apo epsilon 4 allele frequency from 70 AD patients and 106 normal population in Korea. The results suggested that the frequency of epsilon 4 allele among the AD patients (35.7%) was 3 times higher than that among the control population (13.7%). The data, which are in agreement with recent reports, suggests that the apo epsilon 4 allele is associated with AD in Korea.