DNA analysis is a key procedure in genetic engineering. Nowadays, the analysis is often made by PCR with the Taq DNA polymerase. Although the last enzyme price is quite low, the demand for many analyzes leads to many money expenses that are not affordable for many laboratories. In another, many screening tasks do not require highly purified enzyme. Given the unique properties of enzyme that it simplifies its production slightly without resorting to expensive or long techniques such as column chromatography and / or dialysis. Here, the data of the routine use of Taq DNA polymerase prepared according to the protocol developed in our laboratory are presented.
The protocol only takes several hours and does not need qualified personnel or expensive equipment. Yet it gives the preparation of the enzyme adapted to most screening objectives. The isolated Taq DNA polymerase stock can be stored as a suspension of ammonium sulphate in a refrigerator for a long time, at least 6 months. The work enzyme solution is prepared from the on-demand stock suspension, no more than once a month and can also be stored in a refrigerator. The new generation sequencing technology has allowed the detection of rare genetic or somatic mutations and contributed to our understanding of the progression and evolution of the disease.
However, many new generation sequencing technologies first rely on the amplification of DNA, via the reaction of the polymerase chain (PCR), as part of the sample preparation workflow. The errors performed during PCR appear in sequencing data and contribute to false mutations that can define a genetic analysis. In this ratio, a sequencing test at a molecule at a molecule was used to completely catalog the different types of errors introduced during the PCR, including a polymerase interporation, a model switching induced by the structure, a structure. recombination mediated by PCR and DNA damage. In addition to the well-characterized polymerase basic substitution errors, other sources of error have been considered widespread. PCR-mediated recombination per TAQ polymerase has been observed at the single molecule, and surprising would occur as often as basic polymerase substitution errors suggesting that it may be a source of underestimated error for multiplex amplification reactions.
A new method based on fast and fast PCR for genotype mice with a mutation of the leptin receptors (DB / DB mouse).
DB / DB mice are one of the most widely used animal models to study cellular and molecular mechanisms of metabolic disorders, such as diabetes, hyperlipidemia and obesity. The mice carry spontaneous punctual mutations in the gene coding the leptin receptor, resulting in the inactivation of the receptors of the leptin. Since homozygous DB / DB mice are sterile, dB / dB mouse maintenance requires reproduction between heterozygous pairs, which makes genotyping essential to the identification of the offspring. The purpose of this study was to develop a fast and very repeatable method for the genotyping of DB / DB mice, which included only three simple steps: genomic DNA is extracted from third tail or ear notches via a alkaline lysis (approximately20 min);
The samples are then subjected to the reaction of the chain of the chain of the refractory mutation of the tetra-primer-amplification amplification (PCR) using specially designed and validated primer assemblies (~ 17 h); Finally, genotypes are determined by solving PCR products on regular DNA electrophoresis (~ 10 min). The entire DB / DB mouse genotyping procedure can be performed using a regular amplification of the Taq polymerase and PCR within 2 h. The other advantages of this method include high sensitivity and reproducibility. Minimum amounts of mouse tissue are required and genomic samples of the DNA can be stably stored at room temperature for a maximum month. In conclusion, the method is simple, cost-effective, sensitive and reliable, which will greatly facilitate studies using DB / DB mice.
Data of self-made Taq DNA polymerase prepared for screening purposes.
Association of polymorphism of vitamin D receptor genes in patients with type 2 diabetes in the Kashmir Valley.
GOAL
About 1 billion people in various ethnic and elderly groups have vitamin D deficiency. The high prevalence of such a disability is an imperative public health problem, because hypovitaminosis D is an autonomous risk factor for mortality in the mortality. population in general. Beyond bone integrity and calcium homeostasis, it is involved in many physiological and pathological processes. The role of vitamin D in the pathogenesis and prevention of type 2 diabetes mellitus aroused universal interest.
Methods
This case study at the hospital has been designed to study the combination between 25-hydroxy vitamin D (25 [oh] d) levels and polymorphism of vitamin D genes (VDR) with diabetes and to evaluate their roles As factors of risk of diabetes. 100 cases and controls have been taken. 25 (OH) D levels were analyzed by the chemioleneescence method using a Siemens Advia Centaur analyzer. The genomic DNA has been extracted and the Taq-1 and BSM-1 genotyping in the VDR gene has been performed using the reaction of the polymerization chain followed by a restriction fragment length polymorphism (PCR-RFLP) .
RESULTS
(Oh) The levels of patients with diabetes were significantly lower than those of the controls (19.26 ± 0.95 ng / ml against 25.49 ± 1.02 ng / ml; p = 0.001). (OH) The levels of have been found inversely associated with percentages of glyceated hemoglobin in cases (R2 = 0.74). The results suggest that BSM-1 allele and B (G ALLLE) simple nucleotide polymorphisms could be a susceptibility allele for the diabetes of the Cashmiri population.
Recombinant Measles Virus Large Polymerase (58-149)
Description: Recombinant MeV Large Polymerase containing the large polymerase immunodominant regions, 58-149 amino acids was expressed in E. coli and purified by proprietary chromatographic technique.
Description: This product includes all the reagents in E. coli RNA Polymerase Assay Kit (Catalog number RPA100K) plus the enzyme, 33 ul of 100 x E. coli RNAP. It is for 100 assays of E. coli RNA polymerase reactions in a 384-well assay format.
Conclusions
The gene polymorphisms of the VDR appear to be an important genetic determinant in the progression of diabetes. Considering the important predisposition risk factor, we observed that Taq-1 and BSM-1 were strongly associated with Northern Indian diabetes. But requires a subsequent study as a probable genetic risk marker for diabetes.
Jordan
