Single-strand Conformation Polymorphism Analysis Research Articles

Mutation Screening of Her-2, N-ras and Nf1 Genes in Brain Tumor Biopsies.

A deeper understanding of the complex molecular pathology of brain malignancies is needed in order to develop more effective and targeted therapies of these highly lethal disorders. In an effort to further enlighten the molecular pathology of brain oncogenesis involving the her-2 (erbB-2/neu/ngl)/N-ras/nf1 pathway, we screened the genotypes of specimens from various types of brain tumors. The studied specimens included 35 biopsies of four general categories: 13 neuroglial tumors (4 astrocytomas, 2 oligodendrogliomas, 7 glioblastomas multiforme), 14 meningiomas, 3 other nervous system tumors (2 schwannomas, 1 craniopharyngioma) and 5 metastatic tumors (such as lung carcinomas and chronic myelocytic leukemia). Screening for most common mutations in oncogenes her-2, N-ras and tumor suppressor gene nf1 was conducted with molecular hybridization techniques (Southern blotting, dot blot and single-strand conformational polymorphism (SSCP) analysis, respectively), and was confirmed by DNA sequencing. Gene amplification of her-2 was observed in only two cases (6%), namely in one glioblastoma and in one meningioma. Screening of 3 hot spot codons of the N-ras gene (12, 13 and 61) and subsequent DNA sequencing revealed mutations in 19 biopsies encompassing all categories (54%). Screening for mutations in exons of the nf1 gene by SSCP analysis detected a novel nonsense mutation in exon 31 in a unique case of a glioblastoma biopsy (3%) taken from a patient without neurofibromatosis type I. Activated N-ras appears to be a major oncogene in brain oncogenesis, exhibiting the most important role in the her-2/N-ras/nf1 pathway.

Frameshift Mutations in the Mononucleotide Repeats of TAF1 and TAF1L Genes in Gastric and Colorectal Cancers with Regional Heterogeneity.

Initiation of transcription by RNA polymerase II requires TATA-box-binding protein (TBP)-associated factors (TAFs). TAF1 is a major scaffold by which TBP and TAFs interact in the basal transcription factor. TAF1L is a TAF1 homologue with 95% amino acid identity with TAF1. TAF1 is involved in apoptosis induction and cell cycle regulation, but roles of TAF1 and TAF1L in tumorigenesis remain unknown. The aim of this study was to explore whether TAF1 and TAF1L genes were mutated in gastric (GC) and colorectal cancers (CRC). In a public database, we found that TAF1 and TAF1L genes had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with microsatellite instability (MSI). We analyzed the mutations in 79 GC and 124 CRC by single-strand conformation polymorphism analysis and DNA sequencing. In the present study, we found TAF1 frameshift mutations (3.8% of CRC with MSI-H) and TAF1L frameshift mutations (2.9% of GC and 3.8% of CRC with MSI-H). These mutations were not found in stable MSI/low MSI (MSS/MSI-L) (0/90). In addition, we analyzed intratumoral heterogeneity (ITH) of TAF1 and TAF1L frameshift mutations in 16 CRC and found that two and one CRC harbored regional ITH of TAF1 and TAF1L frameshift mutations, respectively. Our data indicate that TAF1 and TAF1L genes harbored not only somatic mutations but also mutational ITH, which together might play a role in tumorigenesis of GC and CRC with MSI-H. Our results also suggest that ultra-regional mutation analysis is required for a comprehensive evaluation of mutation status in these tumors.

Detection, Identification, and Characterization of Phytoplasmas Infecting Apple and Pear Trees in Belarus.

In Samochvalovichi, Belarus, apple and pear tree root samples were examined for the presence of phytoplasmas using a universal 16S rDNA-based PCR assay. Out of 27 tested apple trees, 23 were found to be infected by 'Candidatus Phytoplasma mali' and 46 out of 58 pear trees were positive for the presence of 'Ca. P. pyri.' Species were identified by sequence analysis of the 16S rDNA amplicons. The molecular diversity of the phytoplasma isolates was examined by analysis of an hflB gene using single-strand conformation polymorphism (SSCP) and sequence analysis. Therefore hflB gene amplicons from 'Ca. P. mali' and 'Ca. P. pyri' accessions were cloned after amplification. Screening of 640 cloned hflB fragments by SSCP analysis revealed the presence of eight different profiles for 'Ca. P. mali' and 12 different profiles for 'Ca. P. pyri.' The variants were sequenced and compared in multiple alignments. The nucleic acid homology among the hflB gene fragments ranged between 95.4 and 100.0% and 81.3 to 100.0% for 'Ca. P. mali' and 'Ca. P. pyri,' respectively, indicating a high genetic variability within the species. This is the first report on the occurrence of 'Ca. P. mali' and 'Ca. P. pyri' in Belarusian apple and pear trees and their molecular diversity.

Association of Matrilin-3 Gene Polymorphism with Temporomandibular Joint Internal Derangement

Temporomandibular joint internal derangement (TMJ ID) is a multifactorial complex disease characterised by articular disc degeneration. Matrilin-3 is a cartilage and bone-specific adaptor protein, and amino-acid substitutions in the protein are associated with skeletal diseases and joint disorders. We aimed to detect the variants of Matrilin-3 gene (MATN3) in a TMJ ID case-control group and to investigate the risk association of the detected variants with TMJ ID. A case control study was conducted consisting of 57 unrelated TMJ ID patients (32.7 ± 8.2) and 96 unrelated healthy controls (26.63 ± 3.05) without TMJ ID to look for associations with variants of the MATN3 gene. DNA from individual subjects was extracted and each of the eight exons was amplified by polymerase chain reaction using and analyzed by single-strand conformational polymorphism (SSCP) analysis. SSCP variants were subjected to DNA sequence analysis, which yielded band pattern variations in exon 2 of the gene. We further analyzed exon 2 by DNA sequencing to determine the sequence of these variants. We identified SSCP band patterns variants in exon 2 of the MATN3 gene which upon sequencing revealed a single C to T transition mutation (rs28598872) c.447 C>T (g.11608 C>T). This polymorphism is predicted to result in a synonymous mutation (pAla149 = ). The TT and CT genotypes were more prevalent than the CC genotype in TMJ ID patients compared to the control group with a risk factor of 2.12 (confidence intervals [CI] :0.88-5.08) and 2.0 (CI:0.726-5.508). In addition, TMJ ID patients were divided into two groups as anterior disc displacement with reduction (ADDWR) and anterior disc displacement without reduction (ADDWOR) and compared with the controls. The TT and CT genotypes were more prevalent than the CC genotype in ADDWR patients compared to the control group with a risk factor of 3.85 (CI:0.927-16.048) and 3.75 (1.02-13.786), respectively. We found that, among ADDWR patients, the T allele is a risk factor both in homozygous and heterozygous carriers (p < 0.052, p < 0.036). The results of the study indicate a potential role for the MATN3 rs28598872 polymorphism in the pathogenesis of TMJ ID.

DNA Profile of Pacific White Shrimp, L. vannamei Infected by Taura Syndrome Virus Using Single Strand Conformation Polymorphism (SSCP) Analysis

The shrimp infected by Taura syndrome virus (TSV) can survive until harvesting time. At this phase, the growth of this shrimp is relatively normal but it is referred as TSV carrier. This research is expected to provide a recommendation in effort to prevent the occurrence of TSV infection. Therefore, this study was aimed to evaluate the DNA polymorhism of Pacific white shrimp L. vannamei infected by TSV. A total of 60 samples of L. vannamei were collected from SPF (specific pathogen free)-Florida, F1-healthy, F1-tolerance (it is can life normally after infected by TSV), and F1-moribund (diying after infected by TSV). The samples were analysis using PCR with IQ-2000 KIT to determine the positive infection of samples. DNA profile of white shrimp, L. vannamei was significantly different from SPF Florida, F1-moribund, and F1-healthy shrimp. This expression may control the defense mechanism. This can be ascribed to the reason that shrimp survived from TSV infection had a higher amount of locus (3 loci) that were detected from DNA polymorphism of gene in white shrimp contributed to a gene control that was determined from allele variation. Two important alleles (200 bp and 220 bp) unique may be associated with tolerant trait. Those loci only found in F1-tolerance shrimp. However to prove that further necessary to sequences a nucleotide of three loci as responsible for resistance to TSV infection.

THE EXON 5, 6, 7, 8 OF P53 MUTATIONS IN ORAL SQUAMOUS CELLS CARCINOMA

Genetic instability may underlie the etiology of multistep carcinogenesis. The altered p53 gene observed in tumors may represent the expression of such instability and may allow the accumulation of other gene alterations caused by multiple mechanism. p53 gene is the guardian of the genome, that is why we pay more attention to this gene. In this study, we evaluated the significance of p53 mutation in 55 patient with oral squamous carcinoma. Thirty among them underwent well-differentiated carcinoma, while the remaining 25 patientsunderwent poorly differentiated carcinoma. The mutations were detected by PCR-SSCP (Single strand Conformational Polymorphism) analysis in the region between exon 5 and exon 8. The results indicated that the p53 mutation in exon 5 (40%), exon 6 (28%), exon 7 (24%) and exon 8 (8%) were associated with poorly differentiated carcinoma, whereas mutation in exon 5 (10%), exon 6 (30%), exon 7 (40%) and exon 8 (20%) were associated with well-differentiated carcinoma. These observations suggest that p53 mutation in exon5, 6, and 7 have strong correlation with poorly differentiated in oral squamous carcinoma while well-differentiated level was related with mutation in exon 6,7 and 8.

Molecular characterization of exon 3 of caprine myostatin gene in Marwari goat.

Aim:To estimate genetic variability in exon 3 of caprine myostatin gene in Marwari goats.Materials and Methods:A total of 120 blood samples from unrelated Marwari goats were randomly collected from different villages of Bikaner (Rajasthan), India. Genomic DNA was extracted from whole blood using blood DNA isolation kit (Himedia Ltd.) as per manufacturer’s protocol. The quality of extracted genomic DNA was checked on 0.8% agarose gel. Specifically designed a primer set for caprine myostatin (MSTN) gene (Genebank accession no. DQ167575) was used to amplify the exon 3 region of MSTN gene in Marwari goat. The genetic variability in exon 3 of MSTN gene in Marwari goat was assessed on 8% polyacrylamide gel electrophoresis to detect single strand conformation polymorphism (SSCP) pattern.Results:The exon 3 of MSTN gene in Marwari goat showed two types of conformation patterns on 8% polyacrylamide gel. One of the patterns showed only two bands and was considered as genotype AA, whereas another pattern having an extra band was designated as genotype AB. The frequencies of AA and AB genotype for exon 3 region of MSTN gene were calculated as 0.90 and 0.10, respectively.Conclusion:Low level of polymorphism was observed at exon 3 region of MSTN gene in Marwari goat through SSCP analysis. This information could be utilized in future breeding plan to exploit the unique characteristics of Marwari goat of Rajasthan.

Molecular typing and differences in biofilm formation and antibiotic susceptibilities among Prototheca strains isolated in Italy and Brazil

Bovine mastitis caused by Prototheca is a serious and complex problem that accounts for high economic losses in the dairy industry. The main objective of this study was to identify and characterize at genetic level different Prototheca strains and provide the most complete data about protothecal antibiotic resistance. The study involves 46 isolates from Italian (13 strains) and Brazilian (33 strains) mastitic milk. These strains were identified by multiplex PCR and single strand conformation polymorphism analysis and characterized by randomly amplified polymorphic DNA (RAPD)-PCR. Moreover, biofilm production and antibiotic susceptibility were evaluated. Forty-two strains resulted as Prototheca zopfii genotype 2, whereas 4 isolates could belong to a potential new Prototheca species. The RAPD-PCR, performed with 3 primers (M13, OPA-4, and OPA-18), showed a notable heterogeneity among isolates and grouped the strains according to the species and geographical origin. Biofilm production was species-dependent and P. zopfii genotype 2 strains were classified as strong biofilm producers. In vitro antibiotic susceptibility tests indicated that Prototheca strains were susceptible to antibacterial drugs belonging to aminoglycosides group; the highest activity against Prototheca strains was observed in the case of colistin sulfate, gentamicin, and netilmicin (100% of susceptible strains). It is interesting to note that all the Italian P. zopfii genotype 2 strains showed lower minimum inhibitory concentration values than the Brazilian ones. Nisin showed more efficacy than lysozyme and potassium sorbate, inhibiting 31% of the strains. Results obtained in this study confirmed that RAPD-PCR is a rapid, inexpensive, and highly discriminating tool for Prototheca strains characterization and could give a good scientific contribution for better understanding the protothecal mastitis in dairy herd.

Impact of 3′UTR variation patterns of the KRAS gene on the aggressiveness of pancreatobiliary tumors with the KRAS G13D mutation in a Turkish population

ObjectiveSeveral studies have demonstrated the importance of mutations in codons 12, 13 and 61 and variations in the 3′ untranslated region (3′UTR) of the KRAS gene, frequently observed genetic events in the progression of pancreatobiliary tumors (PBT). However, limited data exist on the clinical effect of these alterations. The aim of the current study was to clarify the frequency of relevant alterations of the 3′UTR regions of the KRAS gene and the effect of KRAS 3′UTR polymorphisms on the prognosis of patients with codon 12, 13 and 61 mutations in a Turkish population with PBT. MethodsCodons 12, 13, and 61 and 3′UTRs of the KRAS gene were screened by single-strand conformation polymorphism (SSCP) analysis and DNA sequencing in 43 patients and 10 controls. Chi-squared and independent sample T tests were used to evaluate the results of the mutation analysis and clinical features of the patients. ResultsWe defined the c.38G > A (rs112445441, p.G13D) (39.54%) mutation and two 3′UTR variations, c.*4066delA (rs560890523) (23.26%) and c.*4065_*4066delAA (rs57698689) (6.98%), in the KRAS gene of Turkish patients. There was a statistically significant relationship between the c.*4066delA (rs560890523) and c.*4065_*4066delAA (rs57698689) variations and invasion and lymph node metastasis status of the patients (p < 0.001). Compared to patients with c.38G > A (rs112445441, p.G13D), patients with c.*4066delA (rs560890523) and c.38G > A (rs112445441, p.G13D) presented more aggressive tumors with highly invasive features. The present study contributes findings regarding the clinical effects of KRAS alterations in PBT. Based on our study, further investigations are required.

Analysis of virus structure variants in before- and after-thermotherapy-treated apple plants

ABSTRACTPre-thermotherapy apple plants and their homologous regenerated virus-infected apple plants were used to analyze the effect of high temperature on the structure of plant virus variants. The target bands of apple chlorotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV), and apple mosaic virus (ApMV) in the plants of the two populations were purified and cloned, and 16 positive clones of each sample were randomly selected for single-strand conformation polymorphism (SSCP) analysis. The results showed that a predominant haplotype could be found in the two populations. The main variant was the same both in the regenerated and homologous treated plants, but the percentages in regenerated plants could be lower than, the same as, or higher than that in pre-thermotherapy plants. Two types could be divided depending on the percentage of the predominant haplotype in the treated plants, diversity (less 70%) and simple (more 80%), respectively. The clones of different haplotype in each isolates were sent to sequence, and the alignment results in accordance with that of the SSCP analysis. All the results indicated that thermotherapy could not affect the predominant variants of virus in treated plants, just change its percentage.

Frameshift Mutations of HSPA4 and MED13 in Gastric and Colorectal Cancers.

Frameshift mutation of genes containing mononucleotide repeats is a feature of gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). In the public genome database, we found that human HSPA4 gene encoding a heats hock protein 70 protein (HSP70-4) and MED13 gene had mononucleotide repeats in the coding sequences that could be targets for frameshift mutation in cancers with MSI. HSP70-4 is a member of HSP70 that is known to play a role in cell survival. MED13 is a member of MED genome-wide transcription regulators that function as a regulator for diverse biological processes. In this study, we analyzed the mutations in 79 GCs and 124 CRCs including high MSI (MSI-H) and microsatellite stable/low MSI (MSS/MSI-L) cases by single-strand conformation polymorphism analysis and DNA sequencing. We found frameshift mutations of HSPA4 gene in two cancers (one GC and one CRC) and MED13 gene in the other two cancers (one GC and one CRC). The frameshift mutations were deletions of one base (c.2396delA (p.Asn799MetfsX50)) in HSPA4 and (c.2175delA (p.Lys725AsnfsX4)) in MED13. Each of HSPA4 and MED13 mutations were detected in GC with MSI-H (1/34: 2.9%) and CRC with MSI-H (1/79: 1.3%), but not in those with MSS. Our data show that unconventional HSPA4 and MED13 genes harbored frameshift mutations in GC and CRC with MSI. These mutations might possibly inactivate their functions and could be a feature of GC and CRC with MSI-H.

Effect of anti-biofouling potential of multi-walled carbon nanotubes-filled polydimethylsiloxane composites on pioneer microbial colonization

In this paper, two carbon nanotube (CNT) nanofillers, namely the multi-walled carbon nanotubes (MWCNTs) and the carboxyl-modified MWCNTs (cMWCNTs), were introduced into the polydimethylsiloxane (PDMS) matrix respectively, in order to produce the PDMS composites with reinforced anti-biofouling properties. The anti-biofouling capacity of the silicone-based coatings, including the unfilled PDMS (P0), the MWCNTs-filled PDMS (PM) and the cMWCNTs-filled PDMS (PC), was examined via the field assays conducted in Weihai, China. The effect of different silicone-based coatings on the dynamic variations of the pioneer microbial-community diversity was analyzed using the single-strand conformation polymorphism (SSCP) technique. The PM and PC surfaces have exhibited excellent anti-biofouling properties in contrast to that of the PDMS surface, with extremely low attachment of the early colonizers, such as juvenile invertebrates, seaweeds and algae sporelings. The PM and PC surfaces can effectively prevent biofouling for more than 12 weeks. These combined results suggest that the incorporation of MWCNTs or cMWCNTs into the PDMS matrix can dramatically reinforce its anti-biofouling properties. The SSCP analysis reveals that compared with the PDMS surfaces, the PM and PC surfaces have strong modulating effect on the pioneer prokaryotic and eukaryotic communities, particularly on the colonization of pioneer eukaryotic microbes. The significantly reduced pioneer eukaryotic-community diversity may contribute to the weakening of the subsequent colonization of macrofoulers.

Association of heat shock protein 70 gene polymorphisms with acute thermal tolerance, growth, and egg production traits of native chickens in Taiwan

Abstract. Heat stress is among the most challenging environmental conditions affecting commercial poultry. It severely affects growth and egg production, particularly in tropical and subtropical regions. This study aimed to examine physiological responses – including triiodothyronine (T3) levels, enzymatic activity of creatine kinase (CK) and lactate dehydrogenase (LDH), respiratory rates, and cloacal temperature – to acute heat stress associated with different genotypes of the HSP70 gene and to evaluate the association of these polymorphisms with growth and egg production. Genotyping was performed by single-strand conformation polymorphism analysis. The polymorphisms identified were A258A, A258G, and G258G. Twenty 12-week old birds were randomly selected from each genotype and exposed to 40 °C ambient temperature for 1 h. Blood samples were collected at 0 and 1 h following heat stress. Respiratory rate and cloacal temperature were measured following 0, 30, and 60 min of exposure. After 1 h, the A258A genotype exhibited lower levels of CK activity and plasma T3. Neither respiratory rate nor cloacal temperature displayed a significant association with the genotypes. Body weight gain differed among the genotypes for males (F = 3.268, P = 0.041) and females (F = 14.029, P &lt; 0.001), and the A258A genotype exhibited the greatest weight gain at 0–16 weeks of age for both genders. There were no significant differences among genotypes regarding egg weight at first egg or the number of eggs laid until 40 weeks of age. The A258A genotype displayed higher heat tolerance with no negative effects on growth performance and egg production.

Endophytic bacteria with antagonistic traits inhabit the wood tissues of grapevines from Tunisian vineyards

Vineyards throughout the world, including Tunisia, are being attacked by Grapevine Trunk Diseases (GTDs) such as Esca and Botryosphaeriae dieback. In this study, the bacterial microflora colonizing the non-necrotic and necrotic wood tissues of Tunisian mature grapevines (cv Muscat d’Italie) was investigated. Both types of tissues were studied in order to decipher microbial communities associated with them and to find a suitable BCA that can be applied to the Tunisian terroir. Single-Strand Conformation Polymorphism (SSCP) analyses showed that complex bacterial communities specifically colonized both types of wood tissues. The 19 most abundant cultivable strains, selected on their morphology, were isolated from plant samples and assigned to Pantoea, Pseudomonas, Curtobacterium and Bacillus species based on the 16S rRNA and rpoB genes. Biochemical and microbiological screenings revealed that those 19 strains (i) metabolized differently carbon sources, even within the same species, (ii) possessed antibiotic genes, (iii) produced siderophores and solubilized phosphates and (iv) had an in vitro antagonistic effect against 3 fungal pathogens (Lasidiodiplodia pseudotheobromae, Neofusicoccum parvum and Schizophyllum commune) involved in GTDs. One strain, Bacillus subtilis “B6”, had a positive effect on young vines of a cultivar, Muscat d’Italie, frequently planted in Tunisia, by reducing the size of the wood necrosis caused by N. parvum, showing its potential to counteract infection caused by this GTDs agent.

Bacterial diversity dynamics of traditional Turkish Ezine Cheese as evaluated by PCR‐DGGE and SSCP analysis

The aim of this study was to evaluate the bacterial ecosystem of milk and Ezine cheese by PCR amplification of the V3 region of the bacterial 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) and by monitoring the bacterial diversity dynamics using PCR single‐strand conformation polymorphism (SSCP) analysis. PCR‐DGGE analysis revealed that 17 different bands and strains belonging to the Lactococcus lactis group and Streptococcus thermophilus were predominant during manufacturing and ripening. SSCP analysis revealed that the bacterial profiles of the two cheese samples were similar.

9. Ovine DRB1 Polymorphism and Its Associations with Body Weight, Milk Contents and Immunological Parameters

Abstract The DRB1 gene is known as a part of the Major Histocompatibility Complex (MHC) with important effects on disease resistance and immunological response in mammals. Single strand conformational polymorphism (SSCP) analysis and direct DNA sequencing methods were employed to study the second exon of DRB1 gene polymorphism in Mehraban and Lori-Bakhtiari sheep, using 155 Mehraban and 167 Lori-Bakhtiari (totally 322) ewes. Eleven different SSCP patterns with 26 SNPs (five new mutations) were found in both breeds. Associations of different genotypes with some traits including body weight, milk contents, milk somatic cell count and some immunological parameters were also studied. The observed genotypes had significant associations (P&lt;0.05) with immunological parameters including serum total protein, α2-globulin, β-globulin, γ-globulin and total globulins and body weight at 3 months of age (weaning). The effects of the genotypes on serum albumin and α1-globulin, body weights at 9 and 12 months of age and milk lactose tended to be significant (P&lt;0.10). The results of the present study indicated that the DRB1 exon 2 is highly polymorphic and could be considered as an important fragment in marker assisted selection, especially for improvement of immunity in sheep.

Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism.

A method based on DNA single-strand conformation polymorphism is demonstrated for effective genotyping of CRISPR/Cas9-induced mutants in rice. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has been widely adopted for genome editing in many organisms. A large proportion of mutations generated by CRISPR/Cas9 are very small insertions and deletions (indels), presumably because Cas9 generates blunt-ended double-strand breaks which are subsequently repaired without extensive end-processing. CRISPR/Cas9 is highly effective for targeted mutagenesis in the important crop, rice. For example, homozygous mutant seedlings are commonly recovered from CRISPR/Cas9-treated calli. However, many current mutation detection methods are not very suitable for screening homozygous mutants that typically carry small indels. In this study, we tested a mutation detection method based on single-strand conformational polymorphism (SSCP). We found it can effectively detect small indels in pilot experiments. By applying the SSCP method for CRISRP-Cas9-mediated targeted mutagenesis in rice, we successfully identified multiple mutants of OsROC5 and OsDEP1. In conclusion, the SSCP analysis will be a useful genotyping method for rapid identification of CRISPR/Cas9-induced mutants, including the most desirable homozygous mutants. The method also has high potential for similar applications in other plant species.

MYOSTATIN GENE ANALYSIS IN THE FIRST GENERATION OF THE BELGIAN BLUE CATTLE IN INDONESIA

A study was conducted to identify the variations of the myostatin and also to evaluate the existence of genetic marker for “double muscling” (11-bp deletion in the third exon of the myostatin gene) in the first generation of Belgian Blue cattle in Indonesia using the Single Strand Conformation Polymorphism (SSCP) and the sequencing analysis. A total of 8 DNA samples belonged to Karya Anugerah Rumpin (KAR) Farm were used in the Single Strand Conformation Polymorphism (SSCP) and the sequencing analysis. There were 3 allele types of myostatin gene based on the SSCP analysis. The Belgian Blue sire has type A allele. The Simmental, Wagyu, SO x BX, Charolais, and the PO cattle have the type B allele, while the Belgian Blue x FH and the Belgian Blue x SO have the type C allele (heterozygous). There are 11-bp deletion in the third exon myostatin gene for the Belgian Blue sire based on the sequencing analysis. The myostatin gene in the Belgian Blue F1 generation individual was heterozygous. This study provides scientific evidence that the 11-bp deletion in the third exon of myostatin gene in the Belgian Blue sire was inherited to its F1 generation (male and female).

DEVELOPMENT AND EVALUATION OF A NOVEL PROBE MICROARRAY FOR GENOTYPING CITRUS TRISTEZA VIRUS USING AN INTEGRATED LAB-ON-CHIP DEVICE

An In-Check system was designed for Citrus tristeza virus (CTV) isolates genotyping. The system includes a set of 44 probes and enables a RT-PCR and microarray hybridization to be handled in a miniaturized silicon lab-on-chip (LoC) device. The 44 probes were designed based on 5'UTR, ORF1a, RdRp, p20, p23 and p33 genes. A probe on the p27 gene was used as a positive control. Capture probes were designed on variable regions of five phylogenetic groups corresponding to T36, T30, VT, NZ-RB, T68 CTV strains and the emerging HA16-5 and NZM16 isolates. Twenty-two isolates collected in Italy, inducing a range of symptoms spanning from vein clearing and slight to strong stem pitting on alemow, to seedling yellows of sour orange, were tested by the LoC device leading to the identification of T30, VT and T30+VT (mixed infections) groups. Eleven out of 23 probes specific for these genotypes reacted producing different patterns among and inside the groups. Results using the LoC device were comparable with capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis and multiple molecular marker (MMM) assays. The preliminary results demonstrate that the device is highly robust and fast.

Identification of a Novel Single Nucleotide Polymorphism in Porcine Beta-Defensin-1 Gene.

Porcine beta-defensin-1 (PBD-1) gene plays an important role in the innate immunity of pigs. The peptide encoded by this gene is an antimicrobial peptide that has direct activity against a wide range of microbes. This peptide is involved in the co-creation of an antimicrobial barrier in the oral cavity of pigs. The objective of the present study was to detect polymorphisms, if any, in exon-1 and exon-2 regions of PBD-1 gene in Large White Yorkshire (LWY) and native Ankamali pigs of Kerala, India. Blood samples were collected from 100 pigs and genomic DNA was isolated using phenol chloroform method. The quantity of DNA was assessed in a spectrophotometer and quality by gel electrophoresis. Exon-1 and exon-2 regions of PBD-1 gene were amplified by polymerase chain reaction (PCR) and the products were subjected to single strand conformation polymorphism (SSCP) analysis. Subsequent silver staining of the polyacrylamide gels revealed three unique SSCP banding patterns in each of the two exons. The presence of single nucleotide polymorphisms (SNPs) was confirmed by nucleotide sequencing of the PCR products. A novel SNP was found in the 5′-UTR region of exon-1 and a SNP was detected in the mature peptide coding region of exon-2. In exon-1, the pooled population frequencies of GG, GT, and TT genotypes were 0.67, 0.30, and 0.03, respectively. GG genotype was predominant in both the breeds whereas TT genotype was not detected in LWY breed. Similarly, in exon-2, the pooled population frequencies of AA, AG, and GG genotypes were 0.50, 0.27, and 0.23, respectively. AA genotype was predominant in LWY pigs whereas GG genotype was predominant in native pigs. These results suggest that there exists a considerable genetic variation at PBD-1 locus and further association studies may help in development of a PCR based genotyping test to select pigs with better immunity.