![]() Three principal components (PCs) described 96.5% of the total variance, and 2 PCs (91%) explained the highest variances. Principal component analysis (PCA) transformed the original data matrix into a number of principal components (PCs). Data pre-treatment via centering and transformation of data by normalization were performed to provide data that are more suitable for analysis and easier to be interpreted. Sixteen amino acids were identified with similar spectral chromatograms. The amino acid compositions of bovine, porcine and fish gelatin were determined by amino acid analysis using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as derivatization reagent. The developed method could be extended for halal authentication of food and pharmaceutical products via detection of porcine gelatin, non-halal gelatin. Conclusion: The chromatograms of LC-MS/MS combined with PCA offered reliable method for differentiation between porcine and bovine gelatins. The loading plot analysis showed that variable of tR32 and m/z32 contributed for the separation of both gelatins. Results: PCA using singvariables retention times (tR) and m/z could successfully classify porcine gelatin and bovine gelatin based on score plots of first principle component (PC1) and second principle component (PC2). The classification between porcine and bovine gelatins was carried out using chemometrics of PCA using retention times and mass to charge ratio (m/z) as variables. Methods: Porcine and bovine gelatins were digested using trypsin enzyme and then subjected to LC-MS/MS analysis. High bloom strength bovine gelatin inhibited amplification of DNA from all three species.Objective: To differentiate porcine gelatin and bovine gelatins using specific peptide markers as determined by liquid chromatography-mass spectrometry tandem with mass spectrometry (LC-MS/MS) and classify both gelatins using retention time and m/z as variables in principal component analysis (PCA). Efficient tomato DNA amplification occurred without low bloom strength bovine gelatin in the reaction mixture. Relative to gelatin-free reactions, addition of low bloom strength bovine derived gelatin improved potato and blueberry DNA amplification, but was dependent on the primer used. Porcine derived gelatins inhibited the PCR or reduced reaction specificity, resulting in poorly resolved major bands and a smear of less abundant products on agarose gels. Furthermore, we show that substitution of bovine serum albumin (BSA) for gelatin increased DNA amplification yields and was required for optimizing the reaction conditions. Technical Abstract: Using a protocol for randomly amplified polymorphic DNA (RAPD) analysis which employs stringent annealing temperatures and relies on a polymerase chain reaction (PCR) buffer containing 1% Triton X-100 and 0.1% gelatin, we have demonstrated using tomato, potato, and blueberry DNA that the addition of the proper type of gelatin to the reaction mixture is important to obtain reliable amplification. Results of this research will assist researchers active in the construction of genetic linkage maps, tagging of desirable genes, fingerprinting cultivars, and conducting population and phylogenetic studies. Furthermore, we show that substitution of bovine serum albumin (BSA) for gelatin increased DNA amplification yields and was superior to gelatin for optimizing reaction conditions. Gelatin derived from porcine skin of high or low gelling strength inhibited the PCR whereas addition of low gelling strength gelatin derived from bovine skin had a positive effect on DNA amplification. Gelatin or bovine serum albumin (BSA) and nonionic detergents are often included in PCR reactions to help stabilize the Taq polymerase enzyme, a key component of the reaction. Using a protocol for RAPD analysis which employs stringent DNA annealing conditions and high levels of detergent and gelatin, we have demonstrated that addition of the proper type of gelatin to the reaction mixture is important to obtain reliable DNA amplification. One of these techniques generates a type of molecular marker termed randomly amplified polymorphic DNA (RAPD). Interpretive Summary: Polymerase chain reaction (PCR) has revolutionized many standard molecular biological techniques.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |