Single-strand Conformation Polymorphism Technique Research Articles

First genetic analysis of Cryptosporidium from humans from Tasmania, and identification of a new genotype from a traveller to Bali.

Little is known about the molecular composition of Cryptosporidium species from humans living in the insular state of Tasmania, Australia. In the present study, we genetically characterized 82 samples of Cryptosporidium from humans following conventional coproscopic testing in a routine, diagnostic laboratory. Using a PCR-coupled single-strand conformation polymorphism (SSCP) technique, targeting portions of the small subunit rRNA (SSU), and 60 kDa glycoprotein (gp60) loci, we identified two species of Cryptosporidium, including C. hominis (subgenotypes IbA10G2, IdA16, IeA12G3T3, and IfA19G1) and C. parvum (IIaA16G1R1 and IIaA18G3), and a new operational taxonomic unit (OTU) that genetically closely resembled C. wrairi. This OTU was further characterized using markers in the actin, Cryptosporidium oocyst wall protein (COWP), and 70 kDa heat shock protein (hsp70) genes. This study provides the first characterization of species and genotypes of Cryptosporidium from Tasmania, and presents clear genetic evidence, using five independent genetic loci, for a new genotype or species of Cryptosporidium in a Tasmanian person with a recent history of travelling to Bali, Indonesia. It would be interesting to undertake detailed molecular-based studies of Cryptosporidium in Indonesia and neighbouring countries, in conjunction with morphological and experimental investigations of new genotypes.

English

The aim of this study was to investigate the phosphoric ore bacterial and eucaryal microbial diversity by using the single strand conformation polymorphism (SSCP) technique and small-subunit (SSU) sequencing. PCR-SSCP patterns showed a remarkably simple microbial community, mainly for bacterial community, but also for the eukaryotic community. According to the highly bacterial variable V3 region of 16S rRNA sequences, five bacterial species were identified: an unidentified species belonged to proteo gamma phylum, an unidentified species belonging to proteo delta phylum, one belonged to Moraxellaceae and two closed to Pseudomonadaceae as predominant bacteria in the mining residue. The study showed for the first time that phosphoric ore harbors major bacterial groups related to organisms having a wide range of environmentally significant functional attributes. These findings provided new opportunities into phosphoric ore microbiology that could be useful in biological system removing waste gases generated from the phosphoric industry.   Key words: Microbial community, bacteria, archaea, eucarya, mining residue.

Association of CDKN2A polymorphisms with the severity of cervical neoplasia in a Brazilian population

Variants of p16(INK4a) and p14(ARF), encoded by the CDKN2A locus, may respond differently to the presence of human papillomavirus (HPV). We investigated the potential association of two CDKN2A polymorphisms, 500C > G (rs11515) and 540C > T (rs3088440), with cervical neoplasia in patients with cervical lesions and healthy controls (n = 492). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), single-strand conformation polymorphism (SSCP) and/or DNA sequencing techniques were employed for genotyping. The 500G allele was found higher, whereas the 540T/T genotype was less frequent in patients with more severe lesions. The CDKN2A variants may have the potential to be markers for the management of patients with cervical neoplasia.

Rabbit MSTN gene polymorphisms and genetic effect analysis.

We analyzed meat samples of nine pure lines of rabbit and its 37 hybrid combinations by sequencing and single-strand conformation polymorphism techniques to explore genetic polymorphisms of all the three exon regions and part of the 5'-regulatory region of the myostatin (MSTN) gene. Thus, we detected a single nucleotide mutation (T→C) on the 476 locus of the 5'-regulatory region, but no mutation sites were detected in the exon areas. The correlation analysis showed that the mutation had some favorable genetic effects, and it resulted in increased liver weight, carcass weight, forelegs weight, back and waist weight, ham weight, and tare weight, whereas it decreased muscle drip loss and cooking loss (P < 0.05). These results suggest that the mutations in the upstream regulatory region of the MSTN gene are beneficial to the rabbit soma development, and the mutations can be used as molecular markers for the selection of the meat quality of rabbits.

DNA polymorphism analysis of Xanthomonas campestris pv. campestris using single-strand conformation polymorphism (SSCP) and random amplified polymorphic DNA (RAPD)

The applicability of the random amplified polymorphic DNA (RAPD) and single-strand conformation polymorphism (SSCP) techniques using M13 and 16S rRNA primers, respectively, for genotyping of the phytopathogenic bacterium Xanthomonas campestris pv. campestris was studied. RAPD provided a simple, rapid, and reliable method to identify genetic variation, and SSCP was used to screen for sequence polymorphisms. A 730-bp region amplified using 16S rRNA primers was subjected to restriction digestion followed by SSCP analysis (restriction fragment length polymorphism (RFLP)-SSCP) to identify polymorphisms, and the results were compared with the RAPD profile obtained using the phage M13 core sequence as a single primer. SSCP is a useful tool for the detection of mutations but large amplicon size can hinder secondary structure formation. Therefore, RAPD using an M13 primer is more efficient for phylogeny detection. The use of a novel polymerase chain reaction (PCR)-based DNA fingerprinting method using both RAPD and SSCP to detect DNA sequence diversity is reported. This approach consists of PCR amplification of the 16S ribosomal DNA with universal primers and analysis of the PCR product by SSCP. This is the first report wherein plant pathogens are subjected to RFLP-SSCP combined with M13 primer-based analysis. This report provides a rapid and reliable method for phylogenetic analysis. Keywords: Xanthomonas campestris pv. campestris, polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP), microbial phylogenetics, randomly amplified polymorphic DNA (RAPD). Afr. J. Biotechnol. Vol. 12 No. 50

Prognostic impact of Wilms tumor gene mutations in Egyptian patients with acute myeloid leukemia with normal karyotype

The Wilms’ tumor (WT1) gene mutations were detected in patients with most forms of acute leukemia. However, the biological significance and the prognostic impact of WT1 mutation in Egyptian patients with acute myeloid leukemia with normal karyotype (AML-NK) are still uncertain. We aimed to evaluate the incidence and clinical relevance of WT1 gene mutations in acute myeloid leukemia with normal karyotype (AML-NK). Exons 7 and 9 of WT1 were screened in samples from 216 adult NK-AML using polymerase chain reaction single-strand conformation polymorphism techniques. Twenty-three patients (10.6%) harbored WT1 mutations. Younger ages and higher marrow blasts were significantly associated with WT1 mutations (P = 0.006 and 0.003 respectively). Complete remission rates were significantly lower in patients with WT1 mutations than those with WT1 wild-type (P = 0.015). Resistance, relapse, and mortality rates were significantly higher in patients with WT1 mutations than those without (P = 0.041, 0.016, and 0.008 respectively). WT1 mutations were inversely associated with NPM1 mutations (P = 0.007). Patients with WT1 mutations had worse disease-free survival (P < 0.001) and overall survival (P < 0.001) than patients with WT1 wild-type. In multivariable analyses, WT1 mutations independently predicted worse DFS (P < 0.001; hazard ratio [HR] 0.036) and overall survival (P = 0.001; HR = 0.376) when controlling for age, total leukocytic count (TLC), and NPM1 mutational status. In conclusion, WT1 mutations are a negative prognostic indicator in intensively treated patients with AML-NK, may be a part of molecularly based risk assessment and risk-adapted treatment stratification of patients with AML-NK.

Germline variants in MRE11/RAD50/NBN complex genes in childhood leukemia

BackgroundThe MRE11, RAD50, and NBN genes encode proteins of the MRE11-RAD50-NBN (MRN) complex involved in cellular response to DNA damage and the maintenance of genome stability. In our previous study we showed that the germline p.I171V mutation in NBN may be considered as a risk factor in the development of childhood acute lymphoblastic leukemia (ALL) and some specific haplotypes of that gene may be associated with childhood leukemia. These findings raise important questions about the role of mutations in others genes of the MRN complex in childhood leukemia. The aim of this study was to answer the question whether MRE11 and RAD50 alterations may be associated with childhood ALL or AML.MethodsWe estimated the frequency of constitutional mutations and polymorphisms in selected regions of MRE11, RAD50, and NBN in the group of 220 children diagnosed with childhood leukemias and controls (n=504/2200). The analysis was performed by specific amplification of region of interest by PCR and followed by multi-temperature single-strand conformation polymorphism (PCR-MSSCP) technique. We performed two molecular tests to examine any potential function of the detected the c.551+19G>A SNP in RAD50 gene. To our knowledge, this is the first analysis of the MRE11, RAD50 and NBN genes in childhood leukemia.ResultsThe frequency of either the AA genotype or A allele of RAD50_rs17166050 were significantly different in controls compared to leukemia group (ALL+AML) (p<0.0019 and p<0.0019, respectively). The cDNA analysis of AA or GA genotypes carriers has not revealed evidence of splicing abnormality of RAD50 pre-mRNA. We measured the allelic-specific expression of G and A alleles at c.551+19G>A and the statistically significant overexpression of the G allele has been observed. Additionally we confirmed the higher incidence of the p.I171V mutation in the leukemia group (7/220) than among controls (12/2400) (p<0.0001).ConclusionThe formerly reported sequence variants in the RAD50 and MRE11 gene may not constitute a risk factor of childhood ALL in Polish population. The RAD50_rs17166050 variant allele is linked to decreased ALL risk (p<0.0009, OR=0.6358 (95%CI: 0.4854-0.8327)). Despite the fact that there is no splicing abnormality in carriers of the variant allele but an excess of the G over the A allele was consistently observed. This data demonstrate that some specific alternations of the RAD50 gene may be associated with childhood ALL.

PD-0005 - Prognostic Biomarkers in a Series of Stage II Colon Cancer

Background: Different clinico-pathological factors are used to define a group of patients with high-risk stage II colon cancer that may benefit from adjuvant chemotherapy. Moreover, several molecular markers, such as microsatellite instability (MSI) and BRAF, have been widely investigated as putative prognostic factors. Recently a high stroma component (tumors with mesenchymal phenotype consistent with epithelial to mesenchymal transition) has also been associated with poor outcome.Methods: Between 1996 and 2006 data from patients diagnosed with colorectal cancer at Hospital Universitari Bellvitge (HUB) and its referral comprehensive cancer center, Institut Català d'Oncologia (ICO)/ L'Hospitalet, were prospectively included in a database. Formalin-fixed paraffin-embedded tissue samples from 432 stage II colon cancer patients operated at HUB were included in the molecular analyses. MSI status, BRAF V600E mutation and tumor-stroma ratio in tumor samples were analyzed. MSI status was assessed by the analysis of 5 mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27). BRAF V600E mutation was analyzed by single strand conformation polymorphism technique. Tumor-stroma ratio was analyzed by immunohistochemistry. Associations between molecular factors and clinical features were assessed by Chi-Squared (X2) tests. A Cox regression model was used to evaluate the Relapse Free Survival (RFS) and the Colon Cancer specific Survival.Results: MSI status could be determined by genotyping in 350 tumor samples. 48 patients (14%) had MSI high (MSI-H) tumors and 302 (86%) had microsatellite stable (MSS) tumor. BRAF status could be determined in 380 tumor samples. 58 cases (15%) were BRAF mutated tumors and 322 (85%) were BRAF wild type tumors. Tumor-stroma ratio was analyzed in 407 cases. 176 tumors (43%) had more than 50% intra-tumor stroma and were classified as stroma-high. MSI-H tumors were significantly located in the right colon (X2 p-value = 1.03e-5), were poorly differentiated (X2 p-value = 0.003) and had more than 12 lymph nodes resected (X2 p-value = 0.037). MSI-H tumors were associated with BRAF mutation (X2 p-value = 0.02) and stroma-low (X2 p-value = 0.0005). When a molecular prognostic risk classification was performed, patients with MSI-H and stroma-low suggested a low risk of relapse comparing to MSI-H/stroma-high, MSS/stroma-low and MSS/stroma-high (Hazard Ratio = 3.15; 95% CI, 0.76 - 13.1, p-value = 0.0602).Conclusion: MSI-H tumors are typically poorly differentiated, located in the right colon and have BRAF mutation as well as low intra-tumor stroma. Our results suggest that low-stroma could partly explain prognosis in patients with MSI-H stage II tumors. Background: Different clinico-pathological factors are used to define a group of patients with high-risk stage II colon cancer that may benefit from adjuvant chemotherapy. Moreover, several molecular markers, such as microsatellite instability (MSI) and BRAF, have been widely investigated as putative prognostic factors. Recently a high stroma component (tumors with mesenchymal phenotype consistent with epithelial to mesenchymal transition) has also been associated with poor outcome. Methods: Between 1996 and 2006 data from patients diagnosed with colorectal cancer at Hospital Universitari Bellvitge (HUB) and its referral comprehensive cancer center, Institut Català d'Oncologia (ICO)/ L'Hospitalet, were prospectively included in a database. Formalin-fixed paraffin-embedded tissue samples from 432 stage II colon cancer patients operated at HUB were included in the molecular analyses. MSI status, BRAF V600E mutation and tumor-stroma ratio in tumor samples were analyzed. MSI status was assessed by the analysis of 5 mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27). BRAF V600E mutation was analyzed by single strand conformation polymorphism technique. Tumor-stroma ratio was analyzed by immunohistochemistry. Associations between molecular factors and clinical features were assessed by Chi-Squared (X2) tests. A Cox regression model was used to evaluate the Relapse Free Survival (RFS) and the Colon Cancer specific Survival. Results: MSI status could be determined by genotyping in 350 tumor samples. 48 patients (14%) had MSI high (MSI-H) tumors and 302 (86%) had microsatellite stable (MSS) tumor. BRAF status could be determined in 380 tumor samples. 58 cases (15%) were BRAF mutated tumors and 322 (85%) were BRAF wild type tumors. Tumor-stroma ratio was analyzed in 407 cases. 176 tumors (43%) had more than 50% intra-tumor stroma and were classified as stroma-high. MSI-H tumors were significantly located in the right colon (X2 p-value = 1.03e-5), were poorly differentiated (X2 p-value = 0.003) and had more than 12 lymph nodes resected (X2 p-value = 0.037). MSI-H tumors were associated with BRAF mutation (X2 p-value = 0.02) and stroma-low (X2 p-value = 0.0005). When a molecular prognostic risk classification was performed, patients with MSI-H and stroma-low suggested a low risk of relapse comparing to MSI-H/stroma-high, MSS/stroma-low and MSS/stroma-high (Hazard Ratio = 3.15; 95% CI, 0.76 - 13.1, p-value = 0.0602). Conclusion: MSI-H tumors are typically poorly differentiated, located in the right colon and have BRAF mutation as well as low intra-tumor stroma. Our results suggest that low-stroma could partly explain prognosis in patients with MSI-H stage II tumors.

Prognostic biomarkers in a series of stage II colon cancer.

3629 Background: Different clinico-pathological factors are used to define a group of patients with high-risk stage II colon cancer (CC) that may benefit of adjuvant treatment. Moreover, several molecular markers, such as microsatellite instability (MSI) and BRAF, have been widely investigated as prognostic factors. Recently a high stroma component (EMT+ subtypes) has also been associated with poor outcome. The aim of the study is to analyze the prognostic value of MSI, BRAF and tumor-stroma ratio in a prospective series of stage II CC patients. Methods: FFPE tissue from 432 stage II CC patients operated at Hospital Universitari de Bellvitge (1996 - 2006) were included in the study. MSI status was assessed by the analysis of 5 mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27). BRAF V600E mutation was analyzed by single strand conformation polymorphism technique. Tumor-stroma ratio was analyzed by immunohistochemistry. Associations between molecular factors and clinical features were assessed by Chi-Squared (X2) tests. A Cox regression model was used to evaluate the Relapse Free (RFS) and the Colon Cancer specific Survival. Results: MSI status could be determined in 350 patients. 48 (14%) had MSI high (MSI-H) tumor. BRAF status could be assessed in 380 cases. 58 (15%) cases were BRAF mutated. Tumor-stroma ratio was analyzed in 407 tumor samples. 176 (43%) tumors had more than 50% intra-tumor stroma and were classified as stroma-high. MSI-H tumors were significantly located in right colon (X2 p-value = 1.03e-5), were poorly differentiated (X2 p-value = 0.003) and had more than 12 lymph nodes resected (X2 p-value = 0.037). MSI-H tumors were associated with BRAF mutation (X2 p-value = 0.02) and stroma-low (X2 p-value = 0.0005). When a molecular prognostic risk classification was performed, patients with MSI-H /stroma-low suggested a low risk of relapse comparing to MSI-H/stroma-high, microsatellite stable (MSS)/stroma-low and MSS/stroma-high patients (HR = 3.15; 95 CI, 0.76 – 13.1, p-value = 0.0602). Conclusions: MSI-H tumors have BRAF mutation as well as low intra-tumor stroma. Our results suggest that tumor-stroma ratio can explain differential prognosis in MSI-H stage II tumors.

Single-Strand Conformation Polymorphism for Molecular Variability Studies of Six Viroid Species

Molecular diversity within six viroid species and different molecular variants, in each species infecting fruit trees was first estimated by the single-strand conformation polymorphism (SSCP) technique and then by direct sequencing analysis. The different variants studied are to three Australian grapevine viroids(AGVd), four citrus dwarfing viroids (CDVd), eleven grapevine yellow speckle viroids type-1 (GYSVd-1), four hop stunt viroids (HSVd), seven peach latent mosaic viroids (PLMVd), and eight pear blister canker viroids (PBCVd). Polyacrylamide gel electrophoresis (PAGE) conditions were compared and optimized to improve the sensitivity of the existing SSCP parameters. The relationships among the various SSCP profiles observed and the variation in nucleotide sequences was studied. The results indicate that the variations of some parameters of electrophoresis for each species allowed higher resolution and hence detection of single nucleotide variations among clones initially clustered into the same group.

Characterization and polymorphism of Keratin Associated Protein 1.4 gene in goats

Keratin-associated proteins (KAPs) are among the main structural components of the animal fibers and form semi-rigid matrix wherein the keratin intermediate filaments (KIFs) are embedded. Variation in the KAP genes has been reported to affect the structure of KAPs and hence fiber characteristics. As no information is available on this gene in Capra hircus therefore, present work was undertaken to characterize and explore the different polymorphic variants of KAP1.4 gene at DNA level in different breeds/genetic groups of goats of Kashmir. Cashmere (Changthangi, 30 animals) and non-Cashmere (Bakerwal and Kargil goats, 20 animals each) goats formed the experimental animals for the study. Single strand conformation polymorphism technique was employed for exploring variability at gene level. On exploring the size variability in KAP1.4 gene between Ovine and Caprine, it was concluded that sheep KAP1.4 gene has a deletion of 30 nucleotides. In comparison to published nucleotide sequences of sheep, goat sequences explored are differing at positions 174, 462 and 568 and at these positions “G”, “T” and “T” nucleotides are present in sheep, but are replaced by “A”, “C” and “C” respectively, in goats. By SSC studies, two genotypes were observed in each genetic group and in Bakerwal goats the genotypes were designated as A1A1 (0.40) and A1A2 (0.60) and were formed by two alleles A1 (0.70) andA2 (0.30). The different SSC patterns observed in Kargil goats were designated as B1B1 (0.35) and B1B2 (0.65) genotypes with frequencies of B1 and B2 alleles as 0.675 and 0.325, respectively. Similarly, two genotypes C1C1 (0.60) and C1C2 (0.40) were observed in Changthangi goats and the frequencies of C1 and C2 alleles were 0.80 and 0.20, respectively. These alleles were later confirmed by sequencing. The sequences of these alleles are available in NCBI under Acc. No's. JN012101.1, JN012102.1, JN000317.1, JN000318.1, JQ436929 and JQ627657. It was concluded that all the alleles observed in a breed were unique to the breed. The designated A1 and A2 alleles of Bakerwal goats differ from each other at positions 245 and the nucleotides observed were “C” or “A” and at position 605 of the nucleotide sequence “T” or “C”, were observed. The designated B1 and B2 alleles of Kargil goats differed from each other at positions 224, 374, 375 and 521. The nucleotides observed in two SSC pattern were C→G, A→G, G→A and T→C, respectively. The designated C1 and C2 alleles of Changthangi goats differed from each other at one position 440 with the change of “A”→“C”.Only two mutations C224G and G375A in Kargil goats resulted in change of the Cysteine (C)→Serine (S) and Alanine (A)→Threonine (T), respectively. The nucleotide sequences of KAP 1.4 gene in Bakerwal, Kargil and Changthangi goats showed 99.7% similarity with each other and 96.7% with sheep and 74.4% with mice. Average guard fiber length and diameter were 81.02±0.16mm and 67.53±0.97μm, respectively, and average down fiber length and diameter was 48.38±0.70mm and 13.32±0.29μm, respectively for Changthangi goats. Average guard fiber length and diameter were 63.51±4.52mm and 105.31±4.48μm, respectively for Bakerwal goats and 62.60±5.03mm and 107.18±2.30μm, respectively for Kargil goats. The effects of the observed genotypes on Cashmere fiber diameter, Cashmere fiber length in Changthangi goats and guard fiber length and guard fiber diameters in Changthangi, Kargil and Bakerwal goats were found to be non-significant (P>0.05).The nonsignificant association between the polymorphism and fiber attributes reported herein may be due to small sample size.

Characterization of the microbial diversity in production waters of mesothermic and geothermic Tunisian oilfields

The microbial diversity of production waters of five Tunisian oilfields was investigated using Single Strand Conformation Polymorphism (SSCP) technique followed by cloning-sequencing. Dynamics of bacterial populations in production waters collected from four wellheads were also evaluated. For all production water samples collected, DNA from Archaea and Eucarya was not sufficiently abundant to permit detection rRNA genes from these groups by PCR-SSCP. In contrast, the bacterial rRNA genes were detected in all samples, except for samples from DOULEB12 and RAMOURA wells. SSCP profiles attested that two of the studied geothermic wells (ASHTART47 and ASHTART48) had shown a clear change over time, whereas a stable diversity was found with the mesophilic DOULEB well (DL3). PCR amplification of rRNA genes was unsuccessful with samples from DOULEB (DL12) at all three sampling time. The bacterial diversity present in production waters collected from pipelines of SERCINA and LITAYEM oilfields was high, while production waters collected from wellheads (ASHTART and DOULEB) exhibited lower diversity. The partial study of the biodiversity showed a dominance of uncultured bacteria and Pseudomonas genus (class of the Gammaproteobacteria) in three of the studied oilfields (ASHTART47, ASHTART48 and DOULEB3). However, for LITAYEM oilfield, a significant dominance of 5 phyla (Proteobacteria, Thermotogae, Firmicutes, Synergistetes, Bacteroidetes) was shown. Our study gives a real picture of the microbiology of some Tunisian oilfield production waters and shows that some of the sequenced bacterial clones have a great similarity to previous sequenced clones described from other oilfields all over the world, indicating that these ecosystems harbour specific microbial communities. These findings can be considered as an indirect indication of the indigenous origin of these clones.

Genotyping of Giardia intestinalis isolated from people living in Sivas, Turkey

The technique of polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) genotyping was used to characterise morphologically identical isolates of Giardia intestinalis from human stool samples. Materials and methods: In this study a total of 17 trophozoite samples, obtained either directly from stool samples or after excystation, or by duodenal aspiration, were used. A set of primers was chosen to amplify the different regions of triose phosphate isomerase (tpi) and a segment of the glutamate dehydrogenase (gdh) genes. A single-stranded conformational polymorphism technique was also used in an attempt to discriminate among some subgroups. Results: Only primers of the 683-bp segment of the tpi gene from the trophozoite samples were suitable for obtaining a PCR product. In the total of 17 trophozoite DNAs where the tpi gene segment was amplified, 9 belonged to assemblage A (53%) and 4 to assemblage B (23.5%). It was not possible to identify assemblages for the remaining 4 samples (23.5%). Conclusion: PCR-RFLP tpi gene application was able to discriminate between G. intestinalis assemblage A and B, but not the other subgroups. Since assemblage A is the more prevalent subgroup compared with assemblage B, this subgroup can be said to be responsible for common Giardia infections in Turkey.

Polymorphisms in the ovine myostatin gene are associated with birth weight but not with weight gain in Iranian Makoei sheep

Myostatin, a transforming growth factor-beta superfamily member, has been well documented as a negative regulator of muscle growth and development. Myostatin, which has 376 amino acids, is synthesized as a precursor protein. Polymorphism of the myostatin gene in Makoei sheep was investigated by PCR and single-strand conformation polymorphism technique (SSCP). Genomic DNA of 92 sheep was isolated from whole blood. A 417-bp myostatin intron I segment was amplified by standard PCR, using locus-specific primers. Four SSCP patterns, representing four different genotypes, were identified. The frequencies of the genotypes were 0.413, 0.293, 0.130, and 0.163 for AD, AC, AE, and BC, respectively. Allele frequencies were 0.4185, 0.0815, 0.2283, 0.2065, and 0.0652 for A, B, C, D, and E, respectively. Observed heterozygosity was 0.7192. There was significant deviation from Hardy-Weinberg equilibrium for this locus. Analysis of myostatin gene sequences revealed heterozygous SNPs, which were in agreement with results obtained in the SSCP analysis. We concluded that SSCP analysis is a quick, sensitive and reliable technique for determination of DNA polymorphisms. The effect of these genotypes on some traits was investigated, and the AD genotype was found to be associated with birth weight. No phenotypic associations were detected with the other genotypes. No associations of myostatin variants with weight gain were detected. We conclude that polymorphism in the ovine myostatin gene is associated with birth weight, but not with weight gain in Iranian Makoei sheep.

Gene-Based Single Nucleotide Polymorphism Markers for Genetic and Association Mapping in Common Bean

BackgroundIn common bean, expressed sequence tags (ESTs) are an underestimated source of gene-based markers such as insertion-deletions (Indels) or single-nucleotide polymorphisms (SNPs). However, due to the nature of these conserved sequences, detection of markers is difficult and portrays low levels of polymorphism. Therefore, development of intron-spanning EST-SNP markers can be a valuable resource for genetic experiments such as genetic mapping and association studies.ResultsIn this study, a total of 313 new gene-based markers were developed at target genes. Intronic variation was deeply explored in order to capture more polymorphism. Introns were putatively identified after comparing the common bean ESTs with the soybean genome, and the primers were designed over intron-flanking regions. The intronic regions were evaluated for parental polymorphisms using the single strand conformational polymorphism (SSCP) technique and Sequenom MassARRAY system. A total of 53 new marker loci were placed on an integrated molecular map in the DOR364 × G19833 recombinant inbred line (RIL) population. The new linkage map was used to build a consensus map, merging the linkage maps of the BAT93 × JALO EEP558 and DOR364 × BAT477 populations. A total of 1,060 markers were mapped, with a total map length of 2,041 cM across 11 linkage groups. As a second application of the generated resource, a diversity panel with 93 genotypes was evaluated with 173 SNP markers using the MassARRAY-platform and KASPar technology. These results were coupled with previous SSR evaluations and drought tolerance assays carried out on the same individuals. This agglomerative dataset was examined, in order to discover marker-trait associations, using general linear model (GLM) and mixed linear model (MLM). Some significant associations with yield components were identified, and were consistent with previous findings.ConclusionsIn short, this study illustrates the power of intron-based markers for linkage and association mapping in common bean. The utility of these markers is discussed in relation with the usefulness of microsatellites, the molecular markers by excellence in this crop.

Mutations in exons of the CYP17-II gene affect sex steroid concentration in male Japanese flounder (Paralichthys olivaceus)

As a specific gene of fish, cytochrome P450c17-II (CYP17-II) gene plays a key role in the growth, development an reproduction level of fish. In this study, the single-stranded conformational polymorphism (SSCP) technique was used to characterize polymorphisms within the coding region of CYP17-II gene in a population of 75 male Japanese flounder (Paralichthys olivaceus). Three single nucleotide polymorphisms (SNPs) were identified in CYP17-II gene of Japanese flounder. They were c.G594A (p.G188R), c.G939A and c.G1502A (p.G490D). SNP1 (c.G594A), located in exon 4 of CYP17-II gene, was significantly associated with gonadosomatic index (GSI). Individuals with genotype GG of SNP1 had significantly lower GSI (P < 0.05) than those with genotype AA or AG. SNP2 (c.G939A) located at the CpG island of CYP17-II gene. The mutation changed the methylation of exon 6. Individuals with genotype AA of SNP2 had significantly lower serum testosterone (T) level and hepatosomatic index (HSI) compared to those with genotype GG. The results suggested that SNP2 could influence the reproductive endocrine of male Japanese flounder. However, the SNP3 (c.G1502A) located in exon 9 did not affect the four measured reproductive traits. This study showed that CYP17-II gene could be a potentially useful candidate gene for the research of genetic breeding and physiological aspects of Japanese flounder.

Allelic polymorphism of Makoei sheep leptin gene identified by polymerase chain reaction and single strand conformation polymorphism

Leptin, a 16-kDa protein secreted from white adipocytes has been implicated in the regulation of food intake, energy expenditure and whole-body energy balance in animals. In the present study, the polymorphism of the leptin gene (LEP) of Makoei sheep was investigated by polymerase chain reaction and single strand conformation polymorphism technique (PCR–SSCP). Genomic DNA was extracted from whole blood samples collected from 130 sheep. A 471 bp LEP exon 3 segment was amplified by standard PCR, using the locus specific primers. PCR products were subjected to SSCP denaturation and polyacrylamide gel electrophoresis. SSCP bands were visualized with silver staining. Five SSCP patterns, representing five different genotypes, were identified. The frequencies of the observed genotypes were 0.17, 0.09, 0.14, 0.37 and 0.23 for AB, BB, AC, BC and CC, respectively. Allele frequencies were 0.15, 0.37 and 0.48 for A, B and C, respectively. The observed heterozygosity (H obs ) value for LEP gene was 0.6769. The chi-square test showed that there was no significant deviation (P>0.01) from Hardy-Weinberg equilibrium for this locus in Makoei sheep population. Key words: Leptin gene (LEP), polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) Makoei sheep.

English

Abstract- The pandemic of metabolic disorders is accelerating in the urbanized world posing huge burden to health and economy. The key pioneer to most of the metabolic disorders is insulin dependent diabetes mellitus or Type 1 Diabetes Mellitus (T1DM) and non-insulin dependent diabetes mellitus commonly known as Type 2 Diabetes Mellitus (T2DM). Both of these forms of diabetes are polygenic and multifactorial. A newly discovered form of diabetes is Maturity Onset Diabetes of the young (MODY). MODY is a monogenic form of diabetes and it is inherited as autosomal dominant disorder. Its age of onsets is at 14-25 years in individuals who have a strong family history of Type 2 Diabetes Mellitus (T2DM). In most of the MODY patients genes involved in mediating and controlling the expression of insulin are concerned. Till date eleven genes that cause MODY have been identified all over the world. But in Pakistan no genetic research has been done on MODY. The aims of the present study were to examine the prevalence and nature of mutation in three common MODY genes on the basis of their occurrence in European populations. These three MODY genes are HNF4-Alpha, GCK, and HNF1-Alpha. 22 members spanning four generations and 11 affected with MODY in Pakistani population were examined. To determine the linkage or exclusion study of the family to six known loci for MODY, a minimum of two micro satellite markers each of the candidate regions of these loci for MODY were genotyped in all the available individuals of this family. To identify genes involved in susceptibility to MODY and to discover new genes and mutations contributing to MODY in Pakistani population, Single Strand Conformation Polymorphism technique (SSCP) was used. Our findings highlighted that those unidentified MODY genes that facilitate to cause this form of diabetes in European population may play a central role for diabetes characterized by autosomal dominant transmission in Pakistani population.

Polymorphism at exon 1 of pit-1 gene and its association with immunocompetence traits in layer chicken

A study was designed to explore genetic variability of partial promoter and exon 1 of pit-1 gene and its effect on immune competence traits in White Leghorn chicken. Single stranded conformation polymorphism technique followed by sequencing was employed to detect polymorphism of the gene. Three genotypes (AA, AB and BB) were observed in White Leghorn chicken where two alleles (A and B) were found. AB genotype and A allele were predominant in the population. Different demographic parameters estimated in the White Leghorn chicken lines indicated the presence of genetic divergence among the lines. The pit-1 mRNA expression varied among the genotypes where genotype BB expressed higher than genotype AA and AB. The genotypes as well as alleles showed significant effect (P < 0.05) on sheep RBC titre in White Leghorn chicken.

Polymorphism analysis of BMPR1B gene by forced RFLP and PCR-SSCP techniques and expression of the mutation in introgressed sheep

The present study was conducted to screen Kashmir valley sheep with history of prolificacy for the presence of FecB mutation. Forced polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single strand conformation polymorphism (SSCP) techniques were employed to detect any polymorphism present in bone morphogenetic protein receptor type 1B (BMPR1B) gene. Further, it was aimed at introgressing the FecB mutation into nonprolific noncarrier sheep. A 140-bp fragment of BMPR1B gene was amplified from isolated genomic DNA and subjected to forced RFLP with restriction enzyme AvaII. Three different RFLP patterns were identified. SSCP analysis showed one-to-one correspondence with RFLP patterns. Sequencing of the samples showing different patterns revealed that the wild (+) and mutant (B) alleles were different by a single nucleotide substitution in the form of A109G from wild to mutant allele. It led to change in amino acid from Glutamine (Q) to Arginine (R) from wild to mutant allele. The mutation was only detected in NARI-Suwarna and their crosses; all Kashmir valley sheep with prolific history lacked it. The + allele was abundant in the studied population. The FecB mutation was introgressed in nonprolific noncarrier sheep by crossing ewes with NARI-Suwarna rams possessing the mutation. First generation crossing produced heterozygous (B+) progeny. Some of the F(1) heterozygous ewes gave birth to twins when mated to unrelated NARI-Suwarna rams. It showed that FecB mutation was successfully expressing in those crosses.