Production and evaluation of egg derived hot start antibodies

BACKGROUND: Hot start can greatly improve specificity, sensitivity and yield of PCR. Non-specific amplification can occur in PCR when reaction mixture is prepared at room temperature, because Taq DNA polymerase is active and the primers can hybridize non-specifically. Hot start Taq DNA polymerases remain inactive at room temperature and are activated after heating at 95°C preventing non-specific amplification. Monoclonal antibodies against Taq DNA polymerase is the first line of reagents used for turn on regular Taq DNA polymerase into Hot start one. The goal of this research was to produce and evaluate Hot Start antibodies derived from chicken eggs. RESULTS: We performed affinity purification of yolk immunoglobulin (IgY) and obtained polyclonal Hot Start antibodies. The yield of specific antibodies was 0.36 mg per egg or 0.2% of total yolk antibodies. The protocol for real time measurement and Hot start IgY activity assessment was developed. We found that Hot start IgY can reversibly block Taq DNA polymerase activity at 50°C and have no negative impact neither on the Taq DNA polymerase activity after denaturation nor on the reverse transcriptase. We estimated that 1.0 µg of Hot start IgY effectively blocks 5 U activity of Taq DNA polymerase. CONCLUSIONS: Egg derived Hot Start polyclonal antibodies are the cheapest source of Hot start antibodies, from one immune egg we can isolate 0.36 mg IgY, this quantity is enough for producing 1800 U activity of Hot start Taq DNA Polymerase.

Development of Nano-Polymerase Chain Reaction and Its Application / 分析化学

Polymerase chain reaction (PCR) has become one of the powerful technique since its invention in 1980s. Nevertheless, PCR technique is still frequently impaired by its low specificity, poor sensitivity, false positive results, etc. Recently, nanomaterials including metal nanoparticles, carbon nanomaterials, quantum dots and nano metal oxide have been added into PCR solution to improve both quality and productivity of PCR. The nanoparticles assisted PCR ( NanoPCR) has received considerable attentions due to its unprecedented sensitivity, selectivity and efficiency. In this view, the mainly used nanoparticles in NanoPCR, including gold nanoparticles, quantum dots, carbon nanomaterials, graphene and metallic oxide, was firstly summarized. And then, the possible mechanisms for highly improved sensitivity and selectivity were discussed. Finally, recent applications of NanoPCR were described.

Unique pattern of mutations in β-thalassemia patients in Western Uttar Pradesh.

CONTEXT: β-thalassemia is one of the most common heterogeneous inherited single gene disorders. The disease results from one or more of 380 different mutations in the β-globin gene. Uttar Pradesh (U.P.) is the most populous state of India, comprising various ethnic groups and Bareilly is one of the largest cities situated in Western U.P. AIMS: To examine the prevalence of five common β-thalassemian mutations: Intervening Sequence IVS 1-5 (c. 92 + 5 G > C), codon 8/9 (c. 27_28insG), codon 41/42 (c. 124_127delTTCT), IVS 1-1 (c. 92 + 1 G > T) and codon 26 G-A (c. 79G > A) in Western U.P. SETTINGS AND DESIGN: Patients attending camps organized by the Thalassemia Society, Bareilly were selected for the study. MATERIALS AND METHODS: A total of 48 blood samples were collected from the patients of transfusion dependent β-thalassemia from July 2011 to May 2012. All the samples were analyzed for five common mutations by using the Amplification Refractory Mutation System (ARMS)-hot start-polymerase chain reaction (PCR) technique. RESULTS: Among the five common mutations prevalent in India, we were able to detect all except codon 26 G-A (c. 79G > A), which is prevalent in northeast India. These four mutations accounted for 58% of the total number of our patients. The IVS 1-5 (G-C) was found to be the most common mutation with a frequency of 46% and the 2 ndmost common mutation was Fr8/9 (+G) with a frequency of 21%. The frequency of other mutations was IVS1-1 (12%) and Cd 41/42 (4%). CONCLUSION: This study provides evidence that the pattern of mutations in Western U.P. is different from the rest of India and even from the neighboring states (Delhi and Punjab). To the best of our knowledge, mutation Fr8/9, the 2ndmost common mutation in our study has never been reported to be so common from anywhere in India. Some mutations, which are prevalent in other regions are absent in our region (mutation for ε-globin). Hence, these findings can be called unique to Western U.P.

Application of Hot Start PCR Method in PCR-based Preimplantation Genetic Diagnosis

PURPOSE: To determine a method to improve the efficacy and accuracy of preimplantation genetic diagnosis (PGD) - polymerase chain reaction (PCR), we compared hot start PCR and conventional multiplex nested PCR. MATERIALS AND METHODS: This study was performed with single lymphocyte isolated from whole blood samples that were obtained from two couples with osteogenesis imperfecta (OI). We proceeded with conventional multiplex nested PCR and hot start PCR in which essential reaction components were physically removed, and we compared the amplification rate, allele dropout rate and nonspecific products. Afterward, we used selective method for PGD. RESULTS: In the two couples, the respective amplification rate were 93.5% and 80.0% using conventional multiplex nested PCR and 95.5% and 92.0% using hot start PCR. The respective mean allele dropout rates for the two couples were 42.0% and 14.0% with conventional multiplex nested PCR and 36.0% and 6.0% with hot start PCR. CONCLUSION: The results demonstrate that the hot start PCR procedure provides higher amplification rates and lower allele dropout rate than the conventional method and that it decreased the nonspecific band in multiplex nested PCR. The hot start method is more efficient for analyzing a single blastomere in clinical PGD.

Detection of Herpes Simplex Virus Type-1 DNA by In Situ Polymerase Chain Reaction

BACKGROUND: Standard solution-phase PCR cannot localize the amplified DNA products in cells or tissue sections. Recently, in situ PCR technique which combines PCR with in situ hybridization was developed and applied to detect target DNA or gene expression in the tissue sections. OBJECTIVE: The purpose of this study was to detect the presence of HSV type-1 DNA in herpes simplex lesions by using hot start PCR in situ hybridization and hot start in situ PCR and to compare the sensitivity and specificity of the two methods. The sensitivity and specificity of multiple overlapping primers and a single primer pair in hot start in situ PCR were also compared. METHODS: We performed hot start PCR in situ hybridization and in situ PCR with multiple overlapping primers, and hot start in situ PCR with a single primer pair in paraffin-embedded, formalin-fixed tissues. RESULTS: HSV type-1 DNA was detected in 4 (80%) of.5 cases of herpes simplex and negative in all cases of herpes zoster, verruca vulgaris, and normal skins. One negative case of herpes simplex could not be detected by HSV type-1 specific primers because it might be caused by HSV type-2. There was no difference in the sensitivity, specificity, and intensity of signals between the three methods. CONCLUSION: Hot start in situ PCR with a single primer pair is a simpler, easier, and more rapid technique for detecting the HSV type-1 DNA in lesional tissue sections with similar sensitivity and specificity than hot start PCR in situ hybridization and hot start in situ PCR using multiple overlapping primers.