Polymerase Chain Reaction-restriction Fragment Length Polymorphism Technique Research Articles

Identification of glucose-6-phosphate dehydrogenase variants by utilizing polymerase chain reaction – Restriction fragment length polymorphism based method

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a widespread genetically inherited enzyme disorder caused by mutation in the G6PD gene located in X chromosome. Although sequencing of the gene is considered as the standard method for detection of mutations, but the process is expensive, laborious, time-consuming and not possible to perform in low resource settings. Thus, the present work was aimed to identify the G6PD variants using Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP) based method among the ethnic groups of malaria endemic Northeastern part of India. STANDARD G6PD Analyzer was utilized for screening to identify the G6PD deficient malarial patients. Molecular analysis of the deficient samples was done using PCR-RFLP technique. A total of 17 variants were explored using different restriction endonucleases. Five variants viz., Orissa (63.8 %), Kalyan-Kerala/Jamnagar/Rohini (9.02 %), Mediterranean (8.3 %), A+ (11.8 %) and Mahidol (1.38 %) were detected among the patients. G6PD Orissa was the major variant among the study population. In malaria endemic regions like Northeast India, a mass screening program for identification of G6PD variants needs to be adopted.

Involvement of p.R72P and PIN3 Ins16bp (TP53) Polymorphisms and the I157T (CHEK2) Mutation in Breast Cancer Occurrence in Burkina Faso

Introduction: The TP53 and CHEK2 genes have been described as breast cancer susceptibility genes and some of their polymorphisms have been associated with an increased risk of breast cancer in certain populations.Aim: The objective of this study was to investigate the p.R72P and PIN3 Ins16bp (TP53) polymorphisms and the I157T (CHEK2) mutation developping of breast cancer. Methods: This case-control study had enrolled 144 participants including 65 cases (breast cancer patients) and 79 controls (women without breast abnormalities) in the city of Ouagadougou in Burkina Faso. The DNA was extracted using the method of “salting out” and the genotyping of polymorphisms was performed by ASO-PCR (Allele Specific Oligonucleotides - Polymerase Chain Reaction), conventional PCR and PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism) techniques. Results: The heterozygous genotype (RP) of the p.R72P polymorphism of TP53 gene was in the majority in cases (73.85%) and controls (73.42%). Regarding to the PIN3 Ins16bp polymorphism of TP53 gene, the homozygous wild type (A1A1) was the most represented in both cases (53.85%) and controls (60.76%). Concerning the I157T mutation of CHEK2 gene, only one (01) patient was homozygous mutant (TT) and no controls had the mutation. This study found no association between these polymorphisms and the risk of breast cancer occurrence (p.R72P (OR=0.96; 95%IC (0.59-1.56); p=0.471), PIN3 Ins16bp (OR= 1.1; 95%IC (0.61-1.98); p=0.420)). Conclusion: This study showed that the P allele of the p.R72P polymorphism and the wild-type allele (A1) of the PIN3 Ins16bp polymorphism were in the majority. The I157T mutation was very rare. These polymorphisms were not associated with the risk of developing breast cancer in this study.

Assessment of the association of CYP1A1 gene polymorphisms with the susceptibility of cervical cancer: A case-control study and meta-analysis

BackgroundCervical cancer (CC) is the second most common type of female malignancy in Bangladesh. Polymorphisms in the CYP1A1 gene have been reported to be associated with CC in different populations. This case-control study with meta-analysis was undertaken to assess the relation of CYP1A1 rs4646903 and rs1048943 polymorphisms with the susceptibility of CC. MethodsA total of 185 CC patients and 220 controls were recruited, and the PCR-RFLP (Polymerase chain reaction-restriction fragment length polymorphism) technique was applied for genotyping. Again, 42 eligible studies (24 with rs4646903 and 18 with rs1048943) were included for meta-analysis, and RevMan 5.3 and the MetaGenyo web-based tool were used. ResultsThe rs4646903 polymorphism was significantly linked with CC in all association models, namely, additive 1, additive 2, dominant, recessive, overdominant, and allele models (OR = 2.41, 4.75, 2.67, 3.61, 2.13, and 2.44 with corresponding 95% CI = 1.55–3.76, 1.81–12.45, 1.75–4.07, 1.39–9.35, 1.38–3.30, and 1.71–3.48, respectively). On the contrary, rs1048943 showed no association (p > 0.05) with CC. Haplotype analysis revealed AT and AC haplotypes significantly decreased (OR = 0.45) and increased (OR = 4.86) CC risk, respectively, and SNPs are in strong linkage disequilibrium (D’ = 0.912, r2 = 0.448). Again, rs4646903 carriers with a contraception history and >5 years of taking contraceptives showed an enhanced risk of CC (OR = 2.39, OR = 3.05). Besides, rs1048943 carriers aged >40 years (OR = 0.44), conceived first child aged ≤18 years (OR = 3.45), and history of contraceptives (OR = 2.18) were significantly linked with CC. Our meta-analysis found that for CYP1A1 rs4646903 codominant 1 (COD 1), codominant 2 (COD 2), codominant 3 (COD 3), dominant model (DM), recessive model (RM), and allele model (AM) in Caucasians and overdominant model (OD) in the overall population are associated with an elevated risk of CC, whereas rs1048943 is also associated with CC in overall, Caucasians and Asians in some genetic models. ConclusionOur case-control study and meta-analysis summarize that CYP1A1 rs4646903 and rs1048943 polymorphisms may be correlated with cervical cancer.

Genetic variation of Limnophora marginata Stein, 1904 (Diptera: Muscidae), associated with an altitudinal gradient in Pedro Moncayo Canton, Pichincha, Ecuador

Abstract. Aguilar KEV, Vivas AYS, Acosta-López C. 2023. Genetic variation of Limnophora marginata Stein, 1904 (Diptera: Muscidae), associated with an altitudinal gradient in Pedro Moncayo Canton, Pichincha, Ecuador. Biodiversitas 24: 269-275. Analyzing heterogeneity in insect populations is relevant to understanding its biology. Thus, we analyzed the genetic variability of two populations of “hunter-flies”, Limnophora marginata Stein, 1904 associated with an altitudinal gradient in the Tocachi and Jerusalem localities, in the Pedro Moncayo Canton, Ecuador, using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of the mitochondrial Cytochrome oxidase I gene. Thirty alleles were detected from analysis with five restriction enzymes; the percentage of polymorphic loci was higher in Tocachi (96.67%) than in Jerusalem (80%). Haplotypes were generated with the corresponding alleles, resulting in a different haplotype for each analyzed individual. Several estimators were used to characterize the population structure. The Analysis of Molecular Variance (AMOVA) evidenced the existence of higher intra-population variability (94%) than inter-population (6%), showing similar information to that found through haplotype analysis. The Fst fixation index indicated that there is no genetic difference between populations (0.056, P<0.05). Nei's genetic distance and Nei's similarity index indicated that L. marginata populations did not show differences between populations. From the results we deduce that the evolutionary force that may be modelling the existent genetic variability may be gene flow, thus establishing a low inter-population genetic structure. The mitochondrial DNA Cytochrome oxidase I gene was an advantageous molecular marker to obtain results that established intra and inter population variability. The characteristics of the mitochondrial DNA were useful to successfully apply the PCR-RFLP technique. Our findings highlight the existence of ecosystem connectivity between L. marginata populations despite altitudinal and habitat differences.

In terms of the PCR-RFLP technique, genetic screening of Ala575Val inactivating mutation in patients with amenorrhea

When mutation is inactivated in the follicle-stimulating hormone receptor (FSHR) gene of patients with amenorrhea, the receptor’s functionality is abolished by completely blocking it, altering the ligand–receptor complex or altering the essential hormone signal transduction. This study aims to detect the frequency and pattern of chromosomal abnormalities and the presence of inactivating mutations (Ala575Val) at position 1540 of the FSHR gene in Iraqi women diagnosed with secondary amenorrhea (SA) and primary amenorrhea (PA). This cross-sectional study was carried out between February and August 2022. Depending on the initial diagnosis of amenorrhea, women were split into two groups: PA and SA. The polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was done for those women with normal karyotyping and hypergonadotropic hypogonadism [follicle stimulating hormone (FSH) > 20 mIU/ml] to determine the genetic cause, such as inactivating mutation in Exon 10E. The FSH of blood in study patients with PA was 72.28 ± 23.60, luteinizing hormone (LH) was 20.60 ± 13.55, and BMI was 24.012 ± 4.06. In SA, the FSH, LH, and BMI values were 69.821 ± 35.95, 16.788 ± 14.12, and 26.12 ± 4.37, respectively. Also, highly significant differences were observed compared with healthy control subjects (P-value < 0.005). The high matching between the results obtained by PCR-RFLP and those obtained by Sanger sequencing techniques used in this study confirmed no detected mutation in Ala575Val at position 1540 in all patients with PA and SA. The study concluded that in the PCR study, Ala575Val encoding genes are highly detected, while in PCR-RFLP, no action of MscI restriction enzyme in position 1540 (region of Ala575Val genotype) has emerged. This gives the impression that women with amenorrhea in the Iraqi population might not have any inactivating mutations in the FSHR gene.

Genetic polymorphism of DGAT1 gene in Mandya and Hassan Sheep of Karnataka

A study was carried out to identify and compare the genetic polymorphism in ovine DGAT1 (Diacylglycerol acyltransferase 1) gene of Mandya and Hassan sheep using PCR-RFLP (polymerase chain reaction – restriction fragment length polymorphism) technique. A total of 100 unrelated animals, 50 each of Mandya and Hassan sheep from farmers’ flocks in the respective breeding tracts were utilized. A 309 bp fragment of ovine DGAT1 gene sequence spanning exons 16–17 and the intervening intron was amplified by PCR employing previously published primers. The RFLP analysis using AluI restriction enzyme, polymorphic pattern revealed two alleles viz., allele C (309 bp fragment) and allele T (272 and 37bp fragments) and three genotypes, CC, CT and TT. The frequencies of CC, CT and TT genotypes were 0.84, 0.16 and 0.00 in Mandya and 0.86, 0.12 and 0.02 in Hassan sheep, respectively. The allelic frequencies for C and T alleles in Mandya and Hassan sheep were 0.92 and 0.08, respectively. Allele C and genotype CC were predominant in both the sheep populations. The sequence analysis revealed two SNPs viz., T->A (transversion) and C->T (transition) at positions 103 and 274 bp, respectively in T allele sequence of ovine DGAT1 gene. BLASTn sequence analysis showed 99% homology with predicted ovine DGAT1 gene (EU178818.1) sequence. Thus, genetic polymorphism was established at exon 1617 locus of ovine DGAT1 gene by PCR-RFLP and two SNPs were identified.

Molecular Detection of Fluoroquinolone Resistance among Multidrug-, Extensively Drug-, and Pan-Drug-Resistant Campylobacter Species in Egypt.

In recent times, resistant foodborne pathogens, especially of the Campylobacter species, have created several global crises. These crises have been compounded due to the evolution of multidrug-resistant (MDR) bacterial pathogens and the emergence of extensively drug-resistant (XDR) and pan-drug-resistant (PDR) strains. Therefore, this study aimed to investigate the development of resistance and the existence of both XDR and PDR among Campylobacter isolates. Moreover, we explored the use of the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique for the detection of fluoroquinolone (FQ)-resistant Campylobacter isolates. A total of 120 Campylobacter isolates were identified depending on both phenotypic and genotypic methods. Of note, cefoxitin and imipenem were the most effective drugs against the investigated Campylobacter isolates. Interestingly, the majority of our isolates (75%) were MDR. Unfortunately, both XDR and PDR isolates were detected in our study with prevalence rates of 20.8% and 4.2%, respectively. All FQ-resistant isolates with ciprofloxacin minimum inhibitory concentrations ≥4 µg/mL were confirmed by the genetic detection of gyrA chromosomal mutation via substitution of threonine at position 86 to isoleucine (Thr-86-to-Ile) using the PCR-RFLP technique. Herein, PCR-RFLP was a more practical and less expensive method used for the detection of FQ resistant isolates. In conclusion, we introduced a fast genetic method for the identification of FQ-resistant isolates to avoid treatment failure through the proper description of antimicrobials.

Penggunaan Teknik Molekuler untuk Mengenali Dermatofita yang Diisolasi dari Hewan Kesayangan di Jakarta dan Bogor

Dermatophytosis is one of the superficial mycoses which causes skin health problems in pet animals. This study conducted molecular characterization using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on specimens obtained from patients suspected of dermatophytosis in several clinics in DKI Jakarta Province and Bogor City. Fifty samples of skin scrapings from patients suspected of clinically dermatophytosis were collected and analyzed by conventional and molecular techniques. The Research aimed to identify dermatophyte that were isolated from pet animals using PCR-RFLP technique. The primers of ITS 1, ITS 4, Chytin Synthase, and cutting enzymes of Dde1 were used in this Research. Four specimens off 50 spesimens were tested positive using direct and culture PCR examination techniques. Based on the sequencing results, Microsporum canis was identified in four spesimens. Specimens that were positive for dermatophytes followed by RFLP using the Dde 1 enzyme. The results of the study showed that molecular techniques were a reliable way to determine the high-precision dermatophytes in diagnosing dermatophytosis. The Results also showed that molecular arrangement of B1 isolate was different from three other isolates.

Discovery of SNPs and indel 11-bp of the myostatin gene and its association with the double-muscled phenotype in Belgian blue crossbred cattle.

Determining double muscle based on the myostatin (MSTN) gene in Belgian blue (BB), Peranakan Ongole (PO) and BB×PO crossbred cattle is very important for crossbreeding programs. This study aimed to investigate single-nucleotide polymorphisms (SNPs) and 11-bp deletions in the coding region of the MSTN gene and their relationship with the double-muscled phenotype in BB×PO crossbred cattle. A total of 86 blood samples were collected from 28 individual BB, 43 individual PO, and 15 individual BB×PO crossbred cattle. SNPs and indel 11-bp variation in the coding region of the MSTN gene were found using the sequencing method, followed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The MSTN gene in the coding region was detected in four SNPs found in PO cattle and its crossbreed. However, the four SNPs could not differentiate normal and double-muscled phenotypes although they are polymorphic. Moreover, in this study, an 11-bp deletion in exon 3 of the MSTN gene in BB cattle was found. In this case, by applying the PCR-RFLP technique using the restriction enzyme NmuCI (Tsp45I) in indel 11-bp, the genotypes that were successfully observed were +/+, +/del.11, and del.11/del.11.

Genetic Identification of all Four Mangrove Mud Crab Species (genus Scylla) Using Multiple Molecular Markers.

Misleading identification and subsequent publications on biological, molecular, and aquaculture data of mangrove mud crab (genus Scylla de Hann 1833) is a major concern in many countries. In this study, multiple molecular markers were used for genetic identification of all four known mud crab species under genus Scylla collected from India, Philippines, Myanmar, Malaysia and Indonesia. Internal Transcribed Spacer (ITS-1), Polymerase chain reaction (PCR)-Restriction Fragment Length Polymorphism (PCR-RFLP) and PCR-based species-specific markers were used to resolve taxonomic ambiguity. PCR-RFLP techniques using NlaIV and BsaJI restriction endonucleases were efficient to differentiate four different mud crab species under genus Scylla with specific fragment profile. The results also justified the use of ITS-1 and PCR-based species-specific markers to identify mud crab species available in many countries quite rapidly and effectively. Several new molecular markers generated during the study are reported here to resolve the taxonomic ambiguity of Scylla species and the results reconfirmed that India is only having two commonly available mud crab species which was reported by the authors earlier.

Association of CTLA4 (rs4553808) and PTPN22 (rs2476601) gene polymorphisms with Hashimoto's thyroiditis disease: A case-control study and an In-silico analysis

In addition to other factors regarding the disease susceptibility, genetic factors are the main causes of Hashimoto's thyroiditis (HT) disease. CTLA4 and PTPN22 are the most prominent genes involved in the immune system regulation, thus effective in the pathogenesis of autoimmune diseases. In the current study, the association of two polymorphisms of CTLA4 and PTPN22 genes, 1661A/G (CTLA-4 gene) and C1858T (PTPN22 gene), with HT was investigated by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) technique. The effects of -1661A/G and C1858T polymorphisms on the mRNA and protein structures of CTLA-4 and PTPN22 were investigated by an in silico analysis. There was no significant difference between the genotype and allele frequencies of PTPN-22 gene polymorphism (rs2476601) in case and control groups. There was a positive association between the frequency of heterozygous genotype of CTLA4-1661A/G polymorphism (rs4553808) and HT (P = .04), but no association was seen regarding allele frequencies. In-silico analysis predicted that the transition of allele A to allele G would lead to the loss of some of the binding sites. Also, the structural analysis of C1858T transition effect on protein revealed a significant influence on PTPN-22 function. Our results showed that heterozygous genotype of -1661A/G CTLA4 gene variation might be related with the risk of HT.

Identification of Bovine Growth Hormone (BGH) Gene Polymorphism Using PCR-RFLP Method in Buffalo Bulls

Growth Hormone (GH) is a single polypeptide chain synthesised and secreted from anterior pituitary gland by somatroph cells. The product of GH gene hastens metabolism and promotes the growth of many organs and tissues especially bone, muscle and visceral organs. It also regulates growth, mammary gland development and lactation. Polymorphism in this gene is associated with increase in growth and development of many tissues in the body.
 Aim: The objective of this study was to investigate the polymorphism of bovine growth hormone (bGH) gene in buffalo bulls (Bubalus bubalis) using the PCR-RFLP (polymerase chain reaction–restriction fragment length polymorphism) technique.
 Design: Genomic DNA was extracted from a total of 10 bulls, consisting of Murrah – Swamp crossbred and pure Swamp buffalo bulls. A The 446 segment of the bGH gene was amplified. The DNA amplicons were detected in 2% agarose gel following 45 minutes of electrophoresis. They were thereafter digesting with AluI endonuclease restriction enzyme, and the digested DNA were detected in 2% agarose gel following electrophoresis for about 45minutes in all samples
 Results: Similar bands of approximately 300 and 146-bp each, with no variation, were detected in 2% agarose gel following electrophoresis in all the animals tested.
 Conclusion: Based on the Alu1 digestion result, all samples produced the same allele of the gene, with no polymorphism detected.

Polymorphism of prolactine genes and its association with body weight in Bayang ducks, local duck from West Sumatera, Indonesia

This study aims to determine the polymorphism of Prolactin (PRL | XbaI and PRL | DraI) genes in Bayang ducks using the PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) technique and its association with weight of ducks aged 1-10 weeks. This study used 200 Bayang duck blood samples consisting of 102 male ducks (♂) and 98 female ducks (♀). DNA from blood samples was isolated using the Genomic DNA Purifification Kit (Promega) using protocol from the manucfacture. The DNA was then amplified using two primers with F: 5′-AAA TTC CCT CTC ACA GTT ACA-3′ and R: 5′-GAT GCA GAG ACA AGT TTC ACC-3′ and F: 5′-GAATAGAACACTTGACCCTG-3′ and R: 5′-TAGAGGAGGCAAGCATAG-3′ which produces fragments with a length of 416 bp and 566 bp. Restriction with XbaI enzymes that recognized the binding site (5-TT CTAGA-3′) resulted 3 genotypes: homozygote (+/+), heterozygote (+/−) and homozygote (−/−) with frequencies 0.455, 0.495 and 0.050 respectively and with frequency allel (+) 0.702 and frequency allel (−) 0.297. While the results of the restriction with enzyme DraI found three types of genotypes, namely (+/+), (+/−) and (−/−) with frequencies of 0.64, 0.35 and 0.01 respectively with frequency allel (+) 0.82 and allele frequencies (−) 0.18. From the results of the analysis, it was found that there was no relationship between these two diversity and weight ducks of duck.

Genetic polymorphisms of the OLR1 and DGAT1 genes associated with milk components in Holstein Friesian dairy cattle under an intensive management in Central Java

Oxidized Low Density Lipoprotein Receptor 1 (OLR1) gene serves to encode the binding vascular surface receptors and to degrade the oxidized low-density lipoprotein. In dairy cattle a nucleotide mutation at 3′UTR location of the OLR1 gene results in C allele of which related to higher milk fat content than A allele. Diacylglycerol O-acyltransferase 1 (DGAT1) gene has a K232A mutation of which K allele also associated with higher milk fat content against A allele. This study was aimed to study the association of variant genotypes of the OLR1 and DGAT1 genes on milk fat and other milk components in Holstein Friesian (HF) dairy cattle. The target of this research was to obtain genotypes of the OLR1 and DGAT1 gene affecting highly to milk fat content and other milk components in domestic HF cattle. Animals observed were Holstein Friesian (HF) lactating cows for 53 heads kept intensively at one government dairy station at BBPTU Baturraden, Central Java. Base mutations at 3′UTR location of the OLR1 gene and at K232A mutation of the DGAT1 gene were identified by PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) techniques. Allele frequency of the base mutation of each gene was analyzed by PopGen 32 program. Milk components of fat, protein, solid non-fat (SNF) and lactose were generated from daily milk test for morning, afternoon and average of both. Study of the association of variant genotypes of the OLR1 and DGAT1 genes on individual milk component was analyzed by GLM considering the fixed effects of lactation period (1-3, 4-6), calving season (1-4 mo., and 5-8 mo.) and calving year (2011, 2012). Duncan Multiple Range Test was used to test significant differences of the averages among subclasses. The OLR1 gene resulted in A allele and C allele of whose the frequencies were respectively 0.457 and 0.543, while the DGAT1 gene resulted in A allele and K allele with the frequencies respectively 0.426 and 0.575. Variant genotypes of the OLR1 gene gave a significant different on milk fat production (P<0.05). Genetic polymorphism of the OLR1 gene had a significant effect on milk fat content in the noon, the highest was for CC genotype, followed by AC, and the lowest was AA, with the fat contents of CC and AC genotypes higher to AA one by 4.87% and 1.38% respectively. However variant genotypes of the OLR1 gene had no significant effect on other milk components (P>0.05). Similarly, variant genotypes of the DGAT1 gene consisting of genotype AK and genotype KK did not significantly affect on all of milk components. It was concluded that there was a fairly good control of the OLR1 gene to fat content in HF cows. The base mutation at the 3′UTR location of the OLR1 gene can be considered as an initial information in developing a genetic assisted selection (GAS) technique for milk fat content in domestic HF cows.

Molecular Typing of Brucella Species Isolated from Humans and Animals Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Technique

Background: Brucellosis is a zoonotic disease that causes major economic and public health problems. It is one of the most important diseases in humans and domestic animals. Hence, the exact identification of Brucella spp. is important for strategies of treatment and control. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is one of the molecular techniques characterized by amplification of deoxyribonucleic acid (DNA) sequence and restriction enzyme digestion. Objectives: This study aimed at identifying genetic polymorphisms of omp2a genes among 90 Brucella isolated from humans and animals, using the PCR-RFLP method. Methods: Ninety Brucella spp. isolated from humans and animals in two different regions of Iran were used in this study. Biochemical tests and the Brucella omp2a (1100 bp) gene-PCR was used for identification of Brucella isolates. Polymerase Chain Reaction products were digested by restriction endonuclease enzyme pstI and gene sequencing analysis was carried out for molecular typing of Brucella strains. Therefore, genetic relatedness was revealed by a dendrogram. Results: Analysis of the 90 Brucella strains by biochemical tests, PCR, and PCR-RFLP methods with PstI enzyme and omp2a sequencing showed four unique RFLP Profiles (P1-P4). Seventy-nine (87.8%) of the Brucella isolates belonged to B. melitensis strain 20236. From 30 animal isolates, nine (30%) belonged to B. melitensis biovare1 and two (6.6%) to B. abortus strain. According to the RFLP dendrogram, group 1 and 2 had higher genetic relatedness similarity. Conclusions: The results showed B. melitensis strain 20236 was the predominant strain among human and animal Brucella isolates. Likewise, according to dendrogram results, the PCR-RFLP technique was not able to separate human and animal species of B. melitensis from B. abortus.

A facile PCR-RFLP method for genotyping of ITPA rs1127354 and rs7270101 polymorphisms.

Inosine triphosphate pyrophosphatase (ITPA) gene single nucleotide polymorphisms (SNPs), rs1127354 and rs7270101, may cause a functional impairment in ITPase enzyme, resulting anemia protection in patients with chronic hepatitis C virus (HCV) infection undergoing ribavirin (RBV)-dependent regimens. The main purpose of this study was to provide and validate a simple, rapid, and inexpensive polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique for genotyping of ITPA rs1127354 and rs7270101 polymorphisms in chronic HCV-infected patients. In the current study, 100 Iranian patients with chronic hepatitis C were examined and genotyped for ITPA rs1127354 and rs7270101 gene polymorphisms. To genotype rs1127354 and rs7270101 polymorphisms, PCR-RFLP technique and sequencing technique were performed on these samples. To validate the PCR-RFLP method, the PCR-RFLP genotyping results should be 100% concordant with the PCR-sequencing results. The rs1127354 and rs7270101 polymorphisms of ITPA gene were genotyped by PCR-RFLP technique and sequencing simultaneously, and the results of both techniques were 100% concordant in all 100 patients. Both PCR-RFLP and sequencing techniques indicated that the genotypic frequency of rs7270101 was 80% AA, 19% AC and 1% CC, and for rs1127354 was 79% CC, 20% CA and 1% AA, respectively. We developed and validated a rapid and inexpensive PCR-RFLP technique for the detection of ITPA rs1127354 and rs7270101 gene polymorphisms.

Association analysis of &lt;i&gt;NUCKS1&lt;/i&gt; and &lt;i&gt;INPP5K&lt;/i&gt; polymorphism with Parkinson’s disease

Genome-wide association studies have reported numerous candidate loci associated with Parkinson's disease (PD). NUCKS1 and INPP5K are two such candidate loci, although they have rarely been reported in Asian populations. To explore these potential genes for PD susceptibility, we investigated the association between PD and two SNPs, rs823114 and rs1109303, located on the NUCKS1 and INPP5K genes, respectively, in the Han population of northern China. We genotyped the two SNPs using the multiplex PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique. A total of 685 subjects including 322 sporadic PD patients and 363 healthy controls were recruited from the population. After Bonferroni correction, our results suggested that there was a significant association of a minor allele (G) in rs823114 with reduced risk of PD development (P = 0.017, OR = 0.768, 95%CI = 0.618 - 0.955), and the difference in genotypes between the PD patients and healthy controls was significant under the dominant model (GA + GG vs. AA). After stratification by gender, males had a lower risk than females (P = 0.008, OR = 0.666, 95%CI = 0.495 - 0.898). However, the distribution of genotype frequency exhibited no significant differences between the PD and control groups (P > 0.025) in INPP5K rs1109303 (P = 0.048, OR = 0.806, 95%CI = 0.650 - 0.998). We conclude that NUCKS1 rs823114 indicates a decreased risk of susceptibility to PD and shows a male genetic distribution bias in the Han Chinese population.

Effect of polymorphisms in the ABCG2, LEPR and SCD1 genes on milk production traits in Holstein cows

This study was performed to investigate the association between polymorphisms in the ABCG2 (ATP-binding cassette sub-family G member 2), LEPR (leptin receptor) and SCD1 (stearoyl-coenzyme A desaturase 1) genes and milk production traits in Holstein dairy cows in Iran. The analysis was conducted on 816 lactations from 408 Iranian Holstein cows. Genotyping was carried out using the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique. The results of the association study revealed that cows with genotype AC of the ABCG2-Y581S single nucleotide polymorphism (SNP) had lower breeding value for milk yield compared with the AA genotype, but showed significantly higher fat and protein percentages. Cows that were homozygous for allele T at the LEPR-T945M locus had higher breeding value for fat yield than those that were homozygous for the C allele. Regarding the SCD1-A293V SNP, cows with genotype AV produced a higher 305-day milk yield in comparison with cows with the VV genotype. Furthermore, cows that were homozygous for allele V showed a significantly higher protein percentage compared with AA and AV genotypes. The results of this study suggest that these SNPs have the potential to be used in programmes based on genomic selection in Iranian dairy herds. ______________________________________________________________________________________ Keywords : Genetic polymorphism, Iranian Holstein, milk fat, milk protein, SNP

Identificação espécie-específica de carnes e produtos cárneos de origem bubalina e bovina pela técnica de PCR-RFLP

Para avaliar a viabilidade da metodologia da Reação em Cadeia da Polimerase associada com o Polimorfismo de Fragmentos de DNA (PCR-RFLP) na identificação de fraude intencional e contaminação acidental em produtos cárneos de origem bubalina, in natura e processados, foram testadas amostras puras e amostras de carnes com misturas controladas, produzidas em laboratório, com adição de 1%, 5%, 10% e 50% de carne bovina em carne de búfalo, homogeneizada crua e em amostras autoclavada. Foram comparados, ainda, diferentes métodos de extração, usando um kit comercial e a técnica clássica, utilizando fenol/clorofórmio. O resultado estatístico foi obtido por tabela de contingência, analisada pelo teste do qui-quadrado (χ2) e do exato de Fisher. A especificidade encontrada foi altamente significativa (P&lt;0,0001). Observou-se também sensibilidade altamente significativa nas diluições a partir de 10% (P&lt;0,0001). A técnica tem alta especificidade e sensibilidade para detectar até mesmo contaminação de 1%, mas a repetibilidade desse resultado impede a aplicação oficial desse método para a inspeção de contaminação acidental, sendo recomendada somente para inspeção de fraude a partir de 10% de substituição. Em carnes autoclavadas, a eficácia do teste é menor. A técnica pode ser empregada para certificação de produto específico (selo de identidade de espécie).

Chloroplast DNA Variations in Wild Brassicas and Their Implication in Breeding and Population Genetics Studies.

Evaluation of chloroplast DNA (cpDNA) diversity in wild relatives of crop brassicas is important for characterization of cytoplasm and also for population genetics/phylogeographic analyses. The former is useful for breeding programs involving wide hybridization and synthesis of alloplasmic lines, while the latter is important for formulating conservation strategies. Therefore, PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) technique was applied to study cpDNA diversity in 14 wild brassicas (including 31 accessions) which revealed a total of 219 polymorphic fragments. The combination of polymorphisms obtained by using only two primer pair-restriction enzyme combinations was sufficient to distinguish all 14 wild brassicas. Moreover, 11 primer pairs-restriction enzyme combinations revealed intraspecific polymorphisms in eight wild brassicas (including endemic and endangered species, B. cretica and B. insularis, resp.). Thus, even within a small number of accessions that were screened, intraspecific polymorphisms were observed, which is important for population genetics analyses in wild brassicas and consequently for conservation studies.