Identification of Myostatin Gene Polymorphism (MSTN|Taq1) Exon-2 in Pitalah Duck Using The PCR-RFLP Method

Association of hypoxia inducible factor-1α polymorphisms with susceptibility to non–small-cell lung cancer

BackgroundHyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS −786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C were calculated using the Hardy Weinberg equation.MethodsThe 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing.ResultsThe allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS −786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele).ConclusionsOur PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants.

Milk protein in dairy cattle consists of casein and whey. Whey in milk of dairy cattle is about 20% with the main component is β-lactoglobulin (7-12%). Polymorphism of the β-lactoglobulin gene affects protein and whey production in milk. Selection at a molecular base to improve whey and protein in milk requires information on genetic diversity of the β-lactoglobulin gene, besides other protein genes. This study was aimed to identify genetic polymorphism of the β-lactoglobulin gene in 88 heads of Friesian Holstein (FH) cows at Cikole Dairy Cattle Breeding and Improvement Station (Cikole DCBIS), Lembang. Genotyping was done using Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) method, and a HaeIII restriction enzyme. Genotype frequency, allele frequency, and heterozygosity value were calculated by Nei method. Hardy-Weinberg equilibrium was calculated by chi-square test. Result showed that the β-lactoglobulin gene in the observed HF cattle was polymorphic, in which there were two alleles (A and B) and three genotypes (AA, AB and BB). Frequencies of the A and B alleles were 0.40 and 0.60 respectively; while those of the AA, AB, and BB genotypes were succesively 0.10; 0.60 and 0.30. Heterozygosity value was obtained for 0.483. The β-lactoglobulin gene was in Hardy - Weinberg equilibrium (χ2cal < χ2table). It is concluded that polymorphic of the β-lactoglobulin gene can be used as an initial information for molecular selection on milk protein composition in FH cows. Key Words : HF Cattle, β-lactoglobuline Gene, Genetic Polymorphism, PCR-RFLP

The pinewood nematode, Bursaphelenchus xylophilus, is the causative agent of pine wilt disease. The virulence of nematodes significantly varies among pine trees, but does not vary within a single pine. To elucidate the reason for no variation in virulence within a single pine, a technique to investigate the population structure of nematodes of different virulence is needed. In this study, the demonstration of interbreeding between virulent and avirulent populations of B. xylophilus in vitro was attempted using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. Ribosomal DNA containing the 5.8S gene, the internal transcribed spacer regions 1 and 2, and partial regions of 18S and 28S genes were used for analysis. First, PCR-RFLP patterns of offspring produced by interbreeding between two individuals of virulent and avirulent isolates were analyzed. The offspring from a single-pair interbreeding showed 3-digested fragment patterns with the restriction enzyme HhaI, patterns the same as the virulent isolate, the avirulent isolate and a hybrid pattern containing both fragments of the virulent and avirulent isolates. The virulent population was mixed with the avirulent one in vitro and propagated nematodes were individually analyzed by PCR-RFLP method. The nematodes showed the same 3 PCR-RFLP patterns as the offspring from a single-pair interbreeding. The detection of nematodes with a hybrid pattern demonstrates the occurrence of interbreeding between virulent and avirulent populations of B. xylophilus.

Rapid Screening Assay for KRAS Mutations by the Modified Smart Amplification Process

To develop a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol using a new genomic marker sequence and a novel set of restriction enzymes in order to detect and discriminate 2 Echinococcus species, E. granulosus and E. multilocularis, found in formalin-fixed paraffin-embedded (FFPE) human tissues. DNA was isolated from 11 FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis. A mitochondrial genomic marker region was amplified and sequenced using a novel primer pair and a new PCR-RFLP protocol was developed for the detection and discrimination of E. granulosus and E. multilocularis using a set of restriction enzymes including AccI, MboI, MboII, and TsoI. The selected marker region was amplified using DNA isolated from FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis and the discrimination of E. granulosus and E. multilocularis was accomplished by use of the novel PCR-RFLP method. In this PCR-RFLP protocol, use of any single restriction enzyme is enough for the discrimination of E. granulosus and E. multilocularis. The PCR-RFLP protocol can be potentially used for the discrimination of 5 other Echinococcus species: E. oligarthus, E. shiquicus, E. ortleppi, E. canadensis, and E. vogeli.

Diabetic kidney disease (DKD) also known as diabetic nephropathy is the leading cause of end-stage renal disease, with multiple genetic and environmental factors involving in its etiology. Angiotensin-converting enzyme (ACE) gene is considered to have an important role in the development and progression of DKD. In this case-control study, we investigated the role of ACE T3892C (rs1800764) polymorphism in the development of DKD in Saudi Arabian population. We recruited 150 type 2 diabetic cases with DKD and 150 type 2 diabetic controls without DKD. The differences in age, sex, systolic blood pressure (SBP), diastolic blood pressure (DBP), duration of diabetes, fasting blood glucose, urinary albumin, albumin/creatinine ratio, serum urea and serum-creatinine between the two groups were analyzed. The genotyping of ACE T3892C polymorphism was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Genotype and allele frequencies were calculated by direct counting. Deviations from Hardy–Weinberg equilibrium (HWE) were tested using the Chi-square (χ2) test in both of studied groups. Odds ratio (OR) with 95% CI were used to evaluate the relationship of ACE T3892C polymorphism with DKD susceptibility. Statistical analysis was performed using SPSS (version 21.0) software and Medcalc software (version 16.4.3). The frequency distribution of ACE T3892C polymorphism was found to be different between case and control groups significantly indicating ACE gene could play an important role in the pathogenesis of DKD in Saudi Arabian population.

This study aims to characterize the phenotype and determine the diversity of the Myostatin (MSTN) gene at Bangkok chickens using the Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP) method. This study used 50 Bangkok chicken blood samples taken from the axillary vein on the wing. DNA was extracted using the protocol Genomic DNA Purification Kit from Promega and then amplified by PCR (polymerase chain reaction) using a pair of primers F: 5'GGT TTT GAC GAC ATG AGC CT3' R: 5'CAG GTG GAA TGT CAT GCA GA3' with product length 955 bp. Amplification products were cut using restriction enzyme MboI with cutting site ↓GATC. MSTN|MboI fragments of the Bangkok chicken were electrophoresed using 2% agarose gel and visualized using doc gel. The average difference test (T-test) on body weight and weight gain of Bangkok chickens from DOC to 3 months by gender. Polymorphism analysis includes allele frequency and genotype. Male and female Bangkok chickens have low phenotype diversity. The MSTN|MboI gene fragment is monomorphic with band positions of 492 bp, 244 bp, and 219 bp resulting in a genotype of ++, and there is one type of allele with a + allele frequency of 100%.

Meat tenderness is an importantquality characteristicfor which consumersare interested. Calpastatin is the calpain inhibitor enzyme and plays an important role in muscle growth and meat quality. The calpastin gene is located on sheep chromosome 5 and its polymorphisms are associated with economic traits. This study was performed to identify calpastatin gene polymorphisms using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Random blood samples were collected from 100 Zel sheep. DNA was extracted by the salting out method and was amplified using PCR. The quantity and quality of the extracted DNA were determined by spectrophotometry and agarose gel electrophoresis. For amplification, a 622 bp fragment exon 1 of the calpastatin gene was used. Amplified fragments were digested by Msp1 restriction enzyme to determine the calpastatin genotypes, and two alleles. M and N were identified. The genotype frequencies of the MM, MN and NN were 0.62, 0.26 and 0.12, respectively in the population under study. Also, the allelic frequency of M and N were 0.75 and 0.25, respectively. Investigation of population genetic structure of Zel sheep revealed that it was not in Hardy-Weinberg equilibrium (p<0.05). The results indicate that it could be useful to consider genetic diversity at calpastatin locus in Zel sheep. Key words: Calpastatin gene, polymorphism, Zel sheep, meat and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

Objective To explore the association between catechol-O-methyltransferase (COMT) Val158Met gene (rs4680) polymorphisms and major depressive disorder (MDD) in Han Chinese. Methods Genotypes and allele frequencies of Val158Met polymorphisms of COMT gene were examined in 250 patients and 300 healthy controls by the method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). t-test, Hardy Weinberg equilibrium and chi-square test were used to conduct statistical analysis. Results There were significant differences between patients and control group in COMT Val158Met genotypes and allele frequency distribution (χ2=20.268, 12.611, P<0.01). Subjects with Met allele had a higher risk than those with Val allele to develop MDD (OR=1.725, 95%CI 1.274-2.335). Conclusion This research suggests that COMT Val158Met polymorphisms are associated with the onset of MDD. Met allele is a risk factor of MDD. Key words: Major depressive disorder; Catechol-O-methyltransferase; Gene polymorphism

Molecular analysis is an easier means to identify desirable genotypes for growth. Candidate gene (s) for growth trait like insulin like growth factor (IGF) has imperative function for growth, body composition, fat deposition, metabolic and skeletal traits and the molecular genetic selection on individual genes is a very efficient method to genetically improve economically important traits in chickens. In the present study, polymorphism of the promoter and 5' untranslated region of IGF-I gene of nativeAseel, was investigated. In order to evaluate the IGF-I gene polymorphism, we used the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Feather samples were collected at random from 50 Aseel chickensof Pakistan. Genomic DNA was extracted using modified salting-out PCI (Phenol: Chlorophorm: Iso-amylacohol) method and amplified by polymerase chain reaction. The promoter and 5' untranslated region of the IGF-I gene was amplified to produce a 621 bp fragment. The PCR products were electrophoresed on 2.5% agarose gel and stained by ethidium bromide and then finally on confirmation of amplification the ampliconswere digested Hinf-I restriction enzyme and this revealed two alleles A and B having all three combinations of genotypes i.e. AA, AB were found. Data were analyzed using Pop Gene 3.1 software package. Allele frequencies (A and B) were 0.53 and 0.47 while genotypic frequency fo AB (66) was significantly higher than AA (20) and AB (66). The Chi-square (χ2) test did not show any deviation from Hardy–Weinberg equilibrium.

Ghrelin (GHRL) is a novel 28-amino acid gut-brain peptide linked to a gene associated with the regulation of growth hormones in birds. This study was carried out to investigate the polymorphism of the Ghrelin gene in Fulani and Yoruba ecotypes chickens in Nigeria. Blood samples were collected from seventy-eight (78) Nigerian indigenous chickens comprising of 41 Yoruba ecotype chickens and 37 Fulani ecotype chickens. Polymerase Chain Reaction-Restriction fragment length polymorphism (PCR-RFLP) method was used and the MboII restriction enzyme cut site 71 of the ghrelin and genetic structure were determined. Population structure was analyzed using allele and genotype frequencies, heterozygosity and genetic variation metrics. Two alleles (C and T) and three genotypes (CC, CT and TT) were observed. In the Yoruba Ecotype, the allele frequencies were C (0.34) and T (0.66) respectively while C (0.45) and T (0.55) were observed in the Fulani ecotype and the overall population was C (0.39) and T (0.61). The genotype frequencies obtained were; in the Yoruba ecotype, CC (0.10), CT (0.48), and TT (0.41) were observed. In the Fulani ecotype, CC (0.22), CT (0.45), and TT (0.32) were also observed, and in the overall population CC (0.15). CT (0.47) and TT (0.37) were observed. FIS values for the Yoruba ecotype (-0.0847) and Fulani ecotype (0.00702) reflects random mating and inbreeding respectively. The effective number of alleles indicates that the Fulani ecotype has more effective alleles compared to the Yoruba ecotype. These results suggest that the Yoruba ecotype may be at Hardy-Weinberg equilibrium, while Fulani ecotype deviates for the ghrelin locus. In summary, our results may open opportunities for genetic improvement in Nigerian indigenous chicken due to the polymorphic nature of the ghrelin gene.

The growth traits were one of the important economic characteristics in beef cattle. One of the growth genes that was suspected as a gene candidate for marker assisted selection (MAS) in Madura cattle was the growth receptor gene (GHR). This study aim was to detect the level of GHR gene polymorphism in Grati-Madura cattle and Pamekasan-Madura cattle populations. A total 86 blood samples of Madura cattle have been collected from the experimental barn of Beef Cattle Research Station and 51 blood samples of Madura cattle from Community Farms in Waru Subdistrict, Pamekasan Madura Regency. Blood samples were isolated using a zymo extraction kit. Detection of GHR growth hormone gene diversity using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method with AluI restriction enzymes. Data analysis used Chi-Square test for genotype, allele frequencies and Hardy Weinberg Equilibrium (HWE). The analysis result showed that GHR genes was detected 3 genotypes namely AA, AG and GG, each with an allele frequency of 0.267, 0.733 and 0.559, 0.441, this shows that the population of Grati-Madura cattle and Pamekasan-Madura cattle were polymorphic with PIC values of 0.315 and 0.372 respectively and were in the moderate category. The observed genotype frequencies in Grati Madura population deviated from HWE, while in Pamekasan madura population was not deviated from HWE. The value of Ho, He PIC and Ne were 0.394, 0.392, 0.315, 1.886 and 0.498, 0.493, 0.372, 1.973 respectively. In conclusion, The GHR gene polymorphism in Grati-Madura cattle and Pamekasan-Madura cattle were polymorphic and very informative so that can be used as gene candidate for MAS.

This study is aimed to determine polymorphism of the Prolactin gene (PRL|PstI) in Sikumbang Jonti ducks using PCR-RFLP (Polymerase Chain Reaction–Restriction Fragment Length Polymorphism) method. This study used 56 Sikumbang Jonti ducks whose blood samples were taken. Gene amplification used a pair of primers forward 5' TGC AAA CCA TAA AAG AAA AGA 3' and reverse 5' CAA TGA AAA GTG GCA AAG CAA 3', which resulted in a 400 bp fragment in exon 5 of the Prolactin (PRL) gene. The amplification product was restricted using the PstI enzyme, which recognizes the truncation site (5' G↓ACGTC 3'). From 56 samples of Sikumbang Jonti ducks identified, just one genotype was found, homozygous (-/-) with only one allele (-). Analysis of the restriction product in Sikumbang Jonti ducks obtained a uniform genetic variation of PRL|PstI (monomorphic) with an allele frequency (-) of 100%.

Genetic factor plays a very important role in determining of cattle performance. A gene which is reported to be controlling cattle growth and body size is pleomorphic adenoma gene 1 (PLAG1). Therefore, the goal of this study was to identify PLAG1 polymorphisms in PO cattle population. A total of 43 unproductive PO cows from the slaughterhouse at Kebumen were used in this study. Blood samples were collected individually when the cattle were slaughtered. Furthermore, DNA genome was harvested from blood samples and used as DNA template for polymerase chain reaction (PCR) targeting g.48308 C>T, g.32212 (19 bp indel) and g.45233 T>C polymorphisms. g.48308 C>T and g.45233 T>C SNPs were genotyped using PCR-restriction fragment length polymorphism (PCR-RFLP) technique and a 19 bp indel of PLAG1 was directly genotyped by observing PCR products. Both allele and genotype frequencies were calculated to test Hardy-Weinberg equilibrium (HWE) status. The results showed that g.48308 C>T SNP and 19 bp indel varied. Three genotypes of the g.48308 C>T SNP namely CC, CT and TT were found in this study and two genotypes (DD and DI) of 19 bp indel were also identified. Moreover, heterozygous CT genotype frequency of g.48308 C>T SNP was 0.51 and T allele frequency was 0.60. In term of 19 bp indel, DD genotype frequency was 0.88 and D allele frequency was 0.94. In conclusion, two polymorphisms of the PLAG1 were identified in PO cattle population.