Polymerase Chain Reaction-single Strand Conformation Polymorphism Research Articles

Comparison of merits of DNA sequencing, PCR-SSCP and MFP assays in the detection of drug resistance in Mycobacterium leprae

Purpose: To compare the merits of conventional Mouse Foot Pad (MFP) assay, Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) analysis, and Direct-DNA sequencing in identification of drug resistance in M. leprae.
 Methods: A total of 41 leprosy cases were investigated for drug resistance using MFP assay, whereby growth results were obtained in 23 cases. The DNAs extracted from these samples were amplified in polymerase chain reaction (PCR) using specific primers in order to determine the drug resistance-loci. Furthermore, PCRSSCP analysis was carried out using PCR amplicons, while gel electrophoresis was performed to identify any shift in mobility in any one of the DNA strands, as well as the pattern of mutation.
 Results: A total of 5 isolates exhibited the highest degree of resistance R100, whereas 4 isolates showed intermediate level of resistance R10. In contrast, the least resistance was seen in R1. There were no mutations in 4 out of 10 strains which were dapsone-resistant, i.e., 1 HR, 2 IR and 1 LR. Moreover, there was no mutation in 305 bp sequence of the rpoB gene. DNA sequencing sensitivity was 60 %, whereas the sensitive isolates tested in the experiments exhibited no mutation, resulting in 100 % specificity.
 Conclusion: These results indicate the advantages of molecular techniques over conventional MFP technique. The study has revealed that PCR-SSCP analysis, a rapid, specific and less expensive method, has greater potential for use in routine testing of the susceptibility of M. leprae to rifampicin and dapsone, than the other tests.

Detection of nucleotide variants in FCGRT (Fc fragment of IgG, receptor, transporter, alpha) gene and their influence on colostral IgG concentration in Indian water buffalo (Bubalus bubalis).

The neonatal Fc receptors (FcRn) mediate the transfer of immunoglobulin G (IgG) molecules from a dam's circulation to the colostrum produced by it immediately after parturition. In ruminants, the calves born are agammaglobulinemic therefore, ingestion of colostrum with high concentration of IgG imparts passive immunity to the newborn. The FcRn molecule is a heterodimer, coded by FCGRT (Fc fragment of IgG Receptor Transporter neonatal) and B2M (Beta 2 microglobulin) genes. Present study attempted to identify single nucleotide polymorphisms (SNPs) in the FCGRT gene in 40 buffaloes of Murrah breed and evaluated the association of these nucleotide variations and haplotypes with IgG concentration in their colostrum. Animals producing colostrum with high IgG and low IgG levels were identified by indirect ELISA and selected. SNPs were detected in the FCGRT gene sequence of selected animals by amplifying it in nine fragments covering all exons (with flanking introns) followed by PCR-single strand conformational polymorphism (PCR-SSCP). A total of nine SNPs were observed of which seven were present in flanking introns and two in exon 4 of the gene. The SNP A75G was non-synonymous and produced an amino acid change from isoleucine to valine. The exonic SNPs and corresponding haplotypes were found to be significantly (P < 0.01 and 0.05 respectively) associated with colostral IgG concentration based on Odds ratios at 95% confidence interval. Polymorphism in FCGRT gene is found to be associated with IgG concentration in colostrum and identification of females with desirable variations may prevent failure of passive transfer in neonatal ruminants.

Association analysis of prolactin and prolactin receptor genes with selected productive and reproductive traits in Egyptian buffalo

A total of 266 records of buffalo raised in two experimental herds in Egypt were assessed to detect prolactin (PRL) and prolactin receptor (PRLR) genes’ polymorphism using PCR-Single Strand Conformational Polymorphism (SSCP) and PCR-Restricted Fragment Length Polymorphism (RFLP) techniques as well as to investigate their association with calf birth weight (BW), weaning weight (WW), lactation period (LP), total milk yield (TMY), stillbirth, calving ease (CE), gestation length (GL), postpartum interval to pregnancy (PPIP), calving interval (CI), and age at first calving (AFC). Predicted breeding values were estimated and used in the association with detected genotypes. A monomorphic pattern of the studied PRL 156 bp segment was recorded and absence of its polymorphism in buffalo was corroborated. We also determined polymorphism of PRLR reflected in three loci: PRLR2, PRLR4, and PRLR9. Significant differences among PRLP9 genotypes (AA, AB, and BB) were displayed for all studied traits as well as among PRLR2 genotypes, except for CE, while PRLR4 genotypes significantly differed only in BW, WW, TMY, stillbirth, GL, and AFC. In practice, strong associations among genotypes of the PRLR gene and the traits of interest candidate this gene to be selective in Egyptian buffalo breeding for improving both productive and reproductive traits.

Mitochondrial mutations in protein coding genes of respiratory chain including complexes IV, V, and mt-tRNA genes are associated risk factors for congenital heart disease.

Most studies aiming at unraveling the molecular events associated with cardiac congenital heart disease (CHD) have focused on the effect of mutations occurring in the nuclear genome. In recent years, a significant role has been attributed to mitochondria for correct heart development and maturation of cardiomyocytes. Moreover, numerous heart defects have been associated with nucleotide variations occurring in the mitochondrial genome, affecting mitochondrial functions and cardiac energy metabolism, including genes encoding for subunits of respiratory chain complexes. Therefore, mutations in the mitochondrial genome may be a major cause of heart disease, including CHD, and their identification and characterization can shed light on pathological mechanisms occurring during heart development. Here, we have analyzed mitochondrial genetic variants in previously reported mutational genome hotspots and the flanking regions of mt-ND1, mt-ND2, mt-COXI, mt-COXII, mt-ATPase8, mt-ATPase6, mt-COXIII, and mt-tRNAs (Ile, Gln, Met, Trp, Ala, Asn, Cys, Tyr, Ser, Asp, and Lys) encoding genes by polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) in 200 patients with CHD, undergoing cardiac surgery. A total of 23 mitochondrial variations (5 missense mutations, 8 synonymous variations, and 10 nucleotide changes in tRNA encoding genes) were identified and included 16 novel variants. Additionally, we showed that intracellular ATP was significantly reduced (P=0.002) in CHD patients compared with healthy controls, suggesting that the mutations have an impact on mitochondrial energy production. Functional and structural alterations caused by the mitochondrial nucleotide variations in the gene products were studied in-silico and predicted to convey a predisposing risk factor for CHD. Further studies are necessary to better understand the mechanisms by which the alterations identified in the present study contribute to the development of CHD in patients.

Identification of Polymorphism within Exon 8 of Bovine HSP90AA1 Gene using PCR-SSCP Technique

Heat shock proteins (HSPs) are a type of molecular chaperones that aid in the recovery of stressed cells and serve as a major system for intracellular self-defense. A study was conducted during the year 2018–19 at College of Veterinary Science Rajendranagar, Hyderabad, Telengana State, India to find polymorphisms in exon 8 of the bovine HSP90AA1 gene in Sahiwal (n=50) and crossbred (n=50) cows. Blood samples were collected from the experimental animals and genomic DNA was isolated. Physiological parameters like body temperature and respiration rate for each animal were taken during the experimental period and the heat tolerance coefficient was calculated. The data on production and reproduction traits were obtained from the history sheets of the animals. To detect the polymorphism, a 539 bp fragment of the HSP90AA1 gene covering exon 8 was subjected to the Polymerase Chain Reaction-Single-Strand Conformation Polymorphism (PCR-SSCP) technique.The PCR-SSCP of exon 8 of HSP90AA1 gene yielded two genotypic patterns AA and AB corresponding to two allelic variants with frequencies of 0.85, 0.15, 0.81 and 0.19 in Sahiwal and crossbred cows, respectively. The PCR-SSCP patterns obtained were correlated with the physiological, productive, and reproductive traits in both Sahiwal and crossbred cows. The association analysis of SSCP patterns of the exon 8 of HSP90AA1 gene revealed non-significant effect in Sahiwal cows, although the AB genotype had a significantly longer service period in crossbred cows.

​Genetic Polymorphism within Exon 3 of HSP90AA1 Gene and its Association Studies in Sahiwal and Crossbred Cows

Background: Heat shock proteins (HSPs) are molecular chaperones that play a critical role in recovering cells from stress and form a primary system for intra cellular self defense. They are highly conserved and play a crucial role in cellular thermo tolerance and heat stress response. Though there are many HSP genes, thermo tolerance is mainly correlated with HSP70 and HSP90 genes in Livestock species. Polymorphisms in these genes have shown an association with heat tolerance, milk production, fertility and disease susceptibility in livestock. They can be used as genetic markers for the selection of animals with better climate resilience, immune response and superior performance. Methods: The present study was carried out in Sahiwal (n=50) and Crossbred cows (n=50) with the objective to identify polymorphisms in HSP90AA1 gene. A 450 bp fragment of bovine HSP90AA1 gene covering exon3 was subjected to Polymerase Chain Reaction-Single-Strand Conformation Polymorphism (PCR-SSCP) technique to identify the polymorphism. PCR-SSCP patterns were correlated with the physiological, productive and reproductive traits in Sahiwal and crossbred cows using the univariate GLM model of SPSS 25. Result: The PCR-SSCP of exon 3 of HSP90AA1 gene yielded two conformational patterns AA and AB corresponding to two allelic variants A and B in both Sahiwal and crossbred cows. The allele frequencies of A and B were 0.78 and 0.22 and 0.84 and 0.16 in Sahiwal and crossbred cows, respectively. The association analysis of SSCP patterns revealed that genotype AA had higher lactation length in Sahiwal cows and higher total lactation milk yield and peak yield in crossbred cows.

Ovine Toll-like Receptor 9 (TLR9) Gene Variation and Its Association with Flystrike Susceptibility.

Simple SummaryFlystrike is a major ectoparasitic disease of sheep and it creates both an economic impact and welfare issue for the sheep industry. Several factors control the responses of sheep to flystrike, and among these, immune response is regarded as an important factor. Toll-like receptors (TLRs) plays a crucial role in the innate immune system by recognizing pathogen-associated molecular patterns derived from various microbes. Of these receptors, TLR9 recognises unmethylated cytosine-phosphate-guanine (CpG) motifs that are known to be prevalent in bacterial genomes and are also reported in Dipteran insects, including Lucilia cuprina, one of the main species associated with flystrike in sheep. In this study, we used a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to investigate TLR9 variation in sheep with and without flystrike, and found that variation in a gene region containing the coding sequence of the putative CpG-DNA binding domain was associated with the likelihood of flystrike occurrence. This suggests that variation in ovine TLR9 may affect a sheep’s response to flystrike.Toll-like receptors (TLRs) are a family of proteins that play a role in innate immune responses by recognising pathogen-associated molecular patterns derived from various microbes. Of these receptors, TLR9 recognises bacterial and viral DNA containing unmethylated cytosine-phosphate-guanine (CpG) motifs, and variation in TLR9 has been associated with resistance to various infectious diseases. Flystrike is a problem affecting the sheep industry globally and the immune response of the sheep has been suggested as one factor that influences the response to the disease. In this study, variation in ovine TLR9 from 178 sheep with flystrike and 134 sheep without flystrike was investigated using a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) approach. These sheep were collected from both commercial and stud farms throughout New Zealand and they were of 13 different breeds, cross-breds and composites. Four alleles of TLR9 were detected, including three previously identified alleles (*01, *02 and *03) and a new allele (*04). In total six single nucleotide polymorphisms (SNPs) were found. Of the three common alleles in the sheep studied, the presence of *03 was found to be associated with a reduced likelihood of flystrike being present (OR = 0.499, p = 0.024). This suggests that variation in ovine TLR9 may affect a sheep’s response to flystrike, and thus the gene may have value as a genetic marker for improving resistance to the disease.

Sequence Variation in the Bovine Lipin-1 Gene (LPIN1) and Its Association with Milk Fat and Protein Contents in New Zealand Holstein-Friesian × Jersey (HF × J)-cross Dairy Cows.

Simple SummaryLipins are a family of proteins involved in lipid metabolism through their phosphatidate phosphatase and transcriptional co-activator activities. Of the three family members identified, lipin-1 is highly upregulated in the bovine mammary gland during lactation, and has been implicated in regulating milk production, albeit with inconsistent results in different breeds of cattle in studies. Accordingly, its role in regulating milk synthesis is not well understood. In this study, we used polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analyses to investigate variation in the bovine lipin-1 gene in New Zealand Holstein-Friesian × Jersey-cross dairy cows and found that variation in the sixth coding exon was associated with milk fat percentage and protein percentage. This suggests that lipin-1 regulates the synthesis of milk fat and milk protein.Lipin-1 is known to play a regulatory role in tissues that function in lipid metabolism. In dairy cows, the lipin-1 gene (LPIN1) is highly expressed in the mammary gland, but its function in milk production is less understood. In this study, we used PCR-single strand conformation polymorphism analysis to investigate sequence variation in three regions of bovine LPIN1 in New Zealand Holstein-Friesian × Jersey (HF × J)-cross dairy cows, including part of the 5′ non-coding region, the region containing the LPIN1β-spliced exon, and the sixth coding exon that encodes the putative transcriptional activating domain of the protein. No variation was found in the LPIN1β-spliced exon, but two sequence variants containing one single nucleotide polymorphism (SNP) were identified in the 5′ non-coding region and four sequence variants containing four non-synonymous SNPs were identified in the sixth coding exon. Among the three common variants of the sixth coding exon, variant C was found to be associated with an increase in milk fat percentage (presence 4.96 ± 0.034% vs. absence 4.81 ± 0.050%; p = 0.006) and milk protein percentage (presence 4.09 ± 0.017% vs. absence 3.99 ± 0.025%; p = 0.001), but no associations (p > 0.01) were detected for milk yield. These results suggest that variation in LPIN1 affect the synthesis of fat and proteins in milk and has potential as a gene-marker to improve milk production traits.

Association of SSCP Polymorphisms of HSP70 Gene with Physiological, Production and Reproduction Performance in Sahiwal and Crossbred Cows

Background: Cellular tolerance to heat stress is mediated by heat shock proteins (HSPs). The HSPs act as molecular chaperones and are transcribed in response to stress. Among different families of these proteins, HSP70 is considered to be related to the development of temperature tolerance. Unraveling polymorphism in heat shock protein genes could be a step towards the identification of genetic markers for selecting heat-tolerant cattle. Methods: The present study was carried out in Sahiwal (n=50) and Crossbred cows (n=50) with the objective to identify polymorphisms in HSP70 gene. Two fragments (295 and 220 bp) of HSP70 gene were subjected to Polymerase Chain Reaction-Single-Strand Conformation Polymorphism (PCR-SSCP) technique. Statistical analysis was performed to study the association of each SSCP genotype on physiological, production and reproduction traits in Sahiwal and crossbred cows using the univariate GLM model of SPSS 25. Result: The PCR-SSCP of 295 bp fragment of HSP70 gene revealed two genotypes AA and AB in Sahiwal cows and two genotypes AA and AC in crossbred cows. The association analysis revealed that genotype AA had higher peak milk yield in Sahiwal cows while the same genotype had higher total lactation milk yield, lower service period and calving interval in crossbred cows. The 220 bp fragment was found to be monomorphic in both Sahiwal and crossbred cows.

Variation in the Ovine Glycogen Synthase Kinase 3 Beta-Interaction Protein Gene (GSKIP) Affects Carcass and Growth Traits in Romney Sheep.

Simple SummaryThe glycogen synthase kinase 3 beta (GSK3β)-interacting protein plays a role in regulating glycogen metabolism, protein synthesis, the cell cycle, and in regulating gene expression. To date, physiological function research into the GSK3β-interacting protein has been focused on cell lines, gene ‘knockout’ models, and over-expression studies, and to our knowledge, there have been no reports on how variation in the GSK3β-interacting protein gene (GSKIP) may affect phenotypic traits. In this study, PCR-SSCP methods were used to screen for variation in exon 1 and exon 2 of GSKIP in 840 New Zealand (NZ) Romney sheep. Two variant sequences were identified in exon 1 and this variation in GSKIP was associated with variation in lamb birth weight, hot carcass weight, and fat depth at the 12th rib.The glycogen synthase kinase 3 beta (GSK3β)-interacting protein (encoded by the gene GSKIP) is a small A-kinase anchoring protein, which complexes with GSK3βand protein kinase A (PKA) and acts synergistically with cAMP/PKA signaling to inhibit GSK3β activity. The protein plays a role in regulating glycogen metabolism, protein synthesis, the cell cycle, and in regulating gene expression. In this study, PCR-single strand conformation polymorphism (PCR-SSCP) analyses were used to screen for variation in exon 1 and exon 2 of GSKIP in 840 New Zealand (NZ) Romney sheep. Two SSCP banding patterns representing two different nucleotide variants (A and B) were detected in an exon 1 region, whereas in an exon 2 region only one pattern was detected. Variants A and B of exon 1 had one non-synonymous nucleotide difference c.37A/G (p.Met13Val). The birthweight of sheep of genotype AA (5.9 ± 0.06 kg) was different (p = 0.023) to sheep of genotype AB (5.7 ± 0.06 kg) and BB (5.7 ± 0.06 kg). The hot carcass weight (HCW) of sheep of genotype AA (17.2 ± 0.22 kg) was different (p = 0.012) to sheep of genotype AB (17.6 ± 0.22 kg) and BB (18.0 ± 0.29 kg), and the fat depth at the 12th rib (V-GR) of sheep of genotype AA (7.7 ± 0.31 mm) was different (p = 0.016) to sheep of genotype AB (8.3 ± 0.30 mm) and BB (8.5 ± 0.39 mm). The results suggest that the c.37A/G substitution in ovine GSKIP may affect sheep growth and carcass traits.

Single-Strand Conformation Polymorphism Fingerprint Method for Dictyostelids.

Dictyostelid social amoebae are a highly diverse group of eukaryotic soil microbes that are valuable resources for biological research. Genetic diversity study of these organisms solely relies on molecular phylogenetics of the SSU rDNA gene, which is not ideal for large-scale genetic diversity study. Here, we designed a set of PCR–single-strand conformation polymorphism (SSCP) primers and optimized the SSCP fingerprint method for the screening of dictyostelids. The optimized SSCP condition required gel purification of the SSCP amplicons followed by electrophoresis using a 9% polyacrylamide gel under 4°C. We also tested the optimized SSCP procedure with 73 Thai isolates of dictyostelid that had the SSU rDNA gene sequences published. The SSCP fingerprint patterns were related to the genus-level taxonomy of dictyostelids, but the fingerprint dendrogram did not reflect the deep phylogeny. This method is rapid, cost-effective, and suitable for large-scale sample screening as compared with the phylogenetic analysis of the SSU rDNA gene sequences.

Identification of sequence variation in the oocyte-derived bone morphogenetic protein 15 (BMP15) gene (BMP15) associated with litter size in New Zealand sheep (Ovis aries) breeds.

Advances in the study of reproductive traits indicate that functional variation in fertility genes may be useful for improving sheep fertility. The aim of this study was to search for variation in the bone morphogenetic protein 15 gene (BMP15) and ascertain any association with litter size in purebred Finnish Landrace sheep (n = 148), Finnish Landrace × Texel-cross sheep (n = 45), and composite sheep (of varying breed background; n = 58) from New Zealand (NZ). A 482bp and 312bp fragment of exon 1 and 2, respectively, of BMP15 were analysed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The additive and dominance effect of BMP15 variation on litter size were estimated using animal and sire models. Two variants (A and B) were detected in exon 1; nosequence variation was detected in exon 2. Variant A had the nucleotide sequence CTT between positions c.31 and c.33, while variant B had a deletion (c.31_33del). The observed frequency for variant A in the Finnish Landrace sheep, Finnish Landrace × Texel-cross sheep and the composite sheep, was 0.77, 0.92, and 0.68, respectively while the frequency of variant B (c.31_33del) was 0.23, 0.08, and 0.32, respectively. An association between litter size and c.31_33del (P < 0.001) was observed in composite sheep. Analysis of more sheep will be required to confirm these results. Litter size did not differ significantly between sheep breeds regardless of the presence/absence of c.31_33del. Results suggested that c.31_33del might be a genetic marker for improving fecundity in some NZ sheep.

Loss of p21ras, p53, and Mdm2 Protein Expression Is Indicative of Worse Prognosis in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) comprise a group of clonal disorders of the hematopoietic stem cell characterized by ineffective hematopoiesis and refractory cytopenias that can progress to acute leukemia. In order to identify molecular events implicated in the pathogenesis of MDS, we analyzed the bone marrow expression and mutational status of ras, p53 and mdm2 genes in a cohort of 25 patients with MDS. The expression of p21ras, p53, and Mdm2 proteins was studied in marrow cytopins stained by APAAP (alkaline phosphatase anti-alkaline phosphatase procedure) using monoclonal antibodies Y13-219, PAb 1801 and 2A10 to detect p21ras, p53 and mdm2 proteins, respectively. N-, K-, H-ras and p53 gene mutations were assessed by PCR-SSCP (polymerase chain reaction/single strand conformation polymorphism) and DNA sequencing to exclude the presence of gene mutations. The quantitative wild-type expression of p21ras, p53 and mdm2 proteins in patient samples (combination of number of cells and staining intensity) was compared to the values of expression of these proteins in 7 normal bone marrow samples. We found overexpression of p21ras and p53 proteins in 12/22 (55%) and 7/23 (30%) of the patients. Five of 7 patients who overexpressed wt-p53 also co-overexpressed wt-p21ras protein. We observed absence of expression of p21ras, p53 and mdm2 in 4/22, 4/23 and 9/19 cases, respectively. Overall survival of patients with absence of p21ras and p53 expression was considerably worse than that of patients with normal expression of these proteins (p=0.0006 to p21ras and p=0.004 to p53 by Log-Rank test; p=0.02 to p21ras and p=0.01 to p53 by Cox proportional hazards model after age, neutrophil and platelet count, and hemoglobin adjustment). We also observed that the patients who exhibited hypoexpression (p=0.055) and absence of mdm2 expression (p=0.07) demonstrated a tendency to have worse prognosis (Log-Rank). There was no correlation between variables p21ras, p53 and mdm2 versus hemoglobin, platelet and neutrophil counts and no association between FAB SMD subcategories and absence of p53 and mdm2 expression by Fisher test (two-tailed), but there was a strong over-representation between absence of p21ras expression and MDS with excess of blasts (p=0.0006). There was a significant correlation between p53 and mdm2 gene expression (p=0.03, Pearson). Only one patient harbored one transversion mutation in codon 12 of K-ras gene and did not correlate with p53 overexpression, but with worse prognosis. No p53 mutation was found. Overall, these findings suggest that the via of signal transduction p21ras/p53 and the auto-regulatory genes p53/mdm2 may be active and play a pivotal role in the pathogenesis of MDS. Hence, loss of bone marrow expression of p21ras, p53 and/or mdm2 proteins is an indicator of worse prognosis in MDS and may serve as early markers of leukemic transformation.

Cloning, characterisation and expression of the SERPINB14 gene, and association of promoter polymorphisms with egg quality traits in layer chicken

ABSTRACT 1. Ovalbumin (SERPINB14) is the most abundant protein present in egg white contributing about 54% of the total egg protein. In this study, the objectives were to clone and characterise the coding sequence of the SERPINB14 gene, to explore its expression profile, identify polymorphisms in the promoter of the gene and explore any association with egg quality traits in White Leghorn chickens. 2. SNPs and mRNA expression of SERPINB14 in White Leghorn chicken lines were detected by PCR-single strand conformation polymorphism (SSCP) along with sequencing and qPCR. The open reading frame (ORF) was cloned in an expression plasmid vector and sequenced. 3. The ORF of this gene was 1161 bp encoding a peptide of 386 amino acids. There were three SNPs observed in the coding region of the gene, one of which was of the mis-sense type, having c562G>A transition which resulted in substitution of alanine to threonine at position 188 in the protein sequence. In both the lines, an increase in expression of the gene was observed after onset of egg production with peak expression at the 40th week of age compared to before onset of lay. The SERPINB14 gene was expressed in the magnum, but not in ovary and infundibulum, tissues of each White Leghorn line. The promoter region of the gene showed SNPs with three haplotypes; H1, H2, and H3. The haplo groups were associated with the egg weight and age at sexual maturity in the IWI line and Haugh unit and albumin index in the IWK line. 4. It was concluded that the ORF of SERPINB14 gene in White Leghorn chicken lines is polymorphic. The promoter region of the gene is also polymorphic and has significant (P < 0.05) association with Haugh unit and egg weight in IWK and IWI chicken lines, respectively.

Nucleotide sequence variation of the major histocompatibility complex class II DQA1 gene in different cattle breeds from Nigeria and New Zealand

The major histocompatibility complex (MHC) plays a role in immune response. Among other activities, the bovine MHC genes (BoLA) trigger immune responses, including the activation of antibody-producing B-cells. In this study, White Fulani (n = 24), Red Bororo (n = 5) and Holstein-White × Fulani-cross (n = 11) cattle from Nigeria, and New Zealand Holstein-Friesian × Jersey-cross (n = 40) cattle were used to investigate variability in exon 2 of BoLA-DQA1. Ten alleles were identified using a PCR-Single Strand Conformation Polymorphism (SSCP) approach and their nucleotide sequences confirmed by DNA sequencing. A total of 12.60 % of all nucleotide positions analysed were revealed to be variable and two novel BoLA-DQA1 alleles are reported here for the first time.

Variation in a Newly Identified Caprine KRTAP Gene Is Associated with Raw Cashmere Fiber Weight in Longdong Cashmere Goats.

Keratin-associated proteins (KAPs) and keratins determine the physical and chemical properties of cashmere fibers as they are the main components of the fibers. It has been reported that ovine KRTAP1-2 affects clean fleece weight, greasy fleece weight and yield in sheep, but the gene has not been described in goats and its effects on fiber traits are unknown. In this study, we identify the keratin-associated protein 1-2 gene (KRTAP1-2) in the goat genome and describe its effect on cashmere fiber traits in 359 Longdong cashmere goats. Six sequence variants (named CAPHI-KRTAP1-2*A to CAPHI-KRTAP1-2*F) were revealed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. These sequences have the highest homology with ovine KRTAP1-2 sequences. There were a 60-bp deletion, a 15-bp insertion and five single nucleotide polymorphisms (SNPs) including two non-synonymous SNPs in the coding sequence. The caprine KRTAP1-2 gene was expressed in the skin tissue, but a signal was not observed for the kidneys, liver, lungs, spleen, heart and longissimus dorsi muscle. Variation in caprine KRTAP1-2 was found to be associated with raw cashmere fiber weight, but not with fiber diameter and length.

Sensitivity and applications of the PCR Single-Strand Conformation Polymorphism method.

PCR Single-Strand Conformation Polymorphism is a method used to identify and detect mutations and is now well known for its many applications on living beings. This paper will discuss the experimental details, limitations and sensitivity of the PCR Single-Strand Conformation Polymorphism method in relation to all existing literature available to us until today. Genomic DNA extraction, PCR amplification and Single-Strand Conformation Polymorphism conditions (concentration of polyacrylamide slab gel electrophoresis, dissociation treatment of double- stranded DNA) and comparison with PCR Restriction Fragment Length Polymorphism are presented. Since its discovery in 1989, there have been many variations, innovations, and modifications of the method, which makes it very easy, safe, fast and for this reason widely applied in clinical diagnostic, forensic medicine, biochemical, veterinary, microbiological, food and environmental laboratories. One of the possible applications of the method is the diagnosis and identification of mutations in new strains of coronaviruses, because science needs more tools to tackle the problem of this pandemic. The PCR Single-Strand Conformation Polymorphism method can be applied in many cases provided that control samples are available and the required conditions of the method are achieved.

Sequence and haplotypes of ankyrin 1 gene (ANK1) and their association with carcass and meat quality traits in yak.

Ankyrin 1 (ANK1) gene has been demonstrated to be a functional candidate gene for meat quality that helps to constitute and maintain the structure of the cell skeleton. In this study, three contiguous ANK1 regions from yak were analyzed using polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP). As a result, nine single-nucleotide polymorphisms (SNPs) were identified, four of them in the coding region and three (c.179 C/A, c.250 G/C, and c.313 C/T) putatively resulting in amino acid changes (p. Ala 60 Glu, p. Asp 84 His, and p. Pro 105 Ser). Some SNPs in promoter region were located within or nearby the putative transcription factor binding sites, such as Sp1 and GATA, which might have an impact on the expression of the yak ANK1 gene. The presence of C1-D3 and C1-A3 were associated with an increased hot carcass weight (p = 0.0045) and a decreased drip loss rate (p = 0.0046). The presence of B1-B3, C1-A3 and C1-D3 had decreased Warner-Bratzler shear force (p = 0.0066, p = 0.0343 and p = 0.0004). The presence of one and two copies of B1-B3 and C1-A3 had decreased Warner-Bratzler shear force (p = 0.0005 and p = 0.0443), and C1-A3 had also decreased drip loss rate (p = 0.0164). These findings indicated that genetic variations of the ANK1 gene would be a preferable biomarker for the improvement of yak meat quality.

Polymorphisms in hormone-sensitive lipase and leptin receptor genes and their association with growth traits in Barki lambs.

Background and Aim:Marker-assisted selection has many advantages over conventional selection in animal breeding. The candidate gene approach has been applied to identify genetic markers associated with economically important traits in livestock. This study was established to investigate variation in the hormone-sensitive lipase (HSL) and leptin receptor (LEPR) genes, and their association with growth traits in Barki lambs.Materials and Methods:Records for birth weight (BW), pre-weaning average daily gain (ADG1), weaning weight (WW), post-weaning average daily gain (ADG2), and marketing weight (MW) were obtained from 247 Barki lambs. Polymerase chain reaction–single-stranded conformational polymorphism analyses were used to detect variation in exon 9 of HSL and exon 19 of LEPR. General linear models were used to test for associations between the variation in ovine HSL and LEPR, and growth traits.Results:The SSCP banding patterns for HSL showed three variants (H1, H2, and H3), which contained two nucleotide-sequence differences (c.1865C>T and c.2038T>C). Two SSCP banding patterns (L1 and L2) were observed for LEPR and these contained two nucleotide-sequence differences (c.2800G>A and c.2978C>G). The HSL genotype showed no effect on the studied traits. The LEPR genotype was proven to have significant effects (p<0.05) on ADG2 and MW. The presence of the L1 variant was associated (p<0.01) with decreased ADG2 and MW.Conclusion:The finding of an association between LEPR gene variation and growth rate after weaning in Barki lambs warrants efforts to improve this trait.

Nucleotide Sequence Variation in the Insulin-Like Growth Factor 1 Gene Affects Growth and Carcass Traits in New Zealand Romney Sheep.

Insulin-like growth factor 1 (IGF1) is a mediator of the effects of growth hormone and polymorphism in the IGF1 gene (IGF1) is reported to affect fat deposition in some livestock species. In this study, nucleotide sequence variation in three regions of ovine IGF1 (part of the 5′ flanking region, the exon 3 region, and the exon 4 region) was investigated in 848 New Zealand Romney lambs using PCR-single strand conformation polymorphism (SSCP) analyses to ascertain if single nucleotide polymorphisms (SNPs) existed. Six SNPs were identified across these three regions. The effect of the sequence variation in the exon 3 and exon 4 regions on growth and carcass traits were investigated. One of the PCR-SSCP sequence variants in the exon 3 region was associated with variation in hot carcass weight, carcass fat depth at the 12th rib measured using video imaging and the percentage proportion of leg lean meat, whereas the other was associated with variation in growth rate to weaning. No associations were detected for the other gene regions analyzed. The results suggest that polymorphism in exon 3 of ovine IGF1 has potential for use as a gene-marker for some carcass and growth traits.