Applications of Eco-Real Time PCR

Eco-Real Time PCR: The Eco Real-Time PCR System offers life science researchers a full-featured real-time PCR system at an attractive price. Its features include: 1. Four-color multiplexing 2. High Resolution Melt (HRM) 3. Fast PCR cycling: 40-cycle PCR in 40 minutes 4. User-friendly, MIQE-compliant software Ecos proprietary technologies provide excellent optical performance along with unmatched temperature control and thermal uniformity for a plate-based format ( 0.1C). Its robust optical system contains two sets of 48 LEDs for a broad range of fluorophores, along with four emission filters and a CCD camera for detection, enabling multiplexing of up to four targets. It is factory-calibrated for SYBR Green I dye, FAM, HEX, VIC, ROX, and Cy5. Eco supports multiple applications, including gene expression quantification and analysis, and genotyping by allele discrimination or high-resolution melt (HRM). The system includes easy-touse data analysis software preloaded on a netbook computer along with other accessories and consumables to provide the best user experience. The software is also provided on a USB drive so that it can be installed on additional computers for convenient access. Real-Time Polymerase Chain Reaction (RT-PCR): Real-Time PCR uses various fluorescent detection chemistries that allow you to monitor the PCR reaction as it progresses. The amount of fluorescent signal generated is directly proportional to the amount of DNA being synthesized during the PCR reaction. Data are collected at each cycle as opposed to traditional PCR, which collects data at the end of the reaction. This allows samples to be characterized by when amplification is first detected as opposed to the amount of product generated after PCR cycling. The greater the amount of the target sequence, the earlier amplification will be detected. One advantage of Real-Time PCR over traditional PCR is that it is a closed-tube system requiring no post-PCR processing. Real-Time PCR has higher precision, increased sensitivity (down to one copy), increased dynamic range (greater than 8 logs), and high resolution (less than two-fold differences). Real-Time PCR (RTPCR) has been used in quantification of gene expression, GMO-testing, viral quantification, validation of array data, pathogen detection, mutation detection and allelic discrimination. RT-PCR for quantification of gene expression (qPCR): The two most commonly used qPCR chemistries are DNA binding dyes and hydrolysis probes (Figure 1). DNA binding dyes fluoresce when bound to double-stranded DNA. Hydrolysis probes fluoresce when the reporter molecule is removed from its quencher molecule by the 5

endonuclease activity of DNA polymerase. Little fluorescence is generated during initial PCR cycles (Figure 2). Data from these early cycles define the baseline for the assay (Phase I). As fluorescence approaches the level of optical detection, the reaction reaches the exponential phase (Phase II), which is the region where the Cq is determined. Cq is the PCR cycle at which the fluorescent signal crosses the detection threshold level and is used for quantification. Finally, as reaction components are consumed and amplicons become abundant, the generation of additional fluorescent signal slows down and eventually reaches a reaction plateau (Phase III).

This information of samples can be compared with the known standards to know the exact level of gene expression in the sample. Eco-RTPCR is compatible for all the chemistries of RTPCR used for this analysis. qPCR has following applications: 1. 2. 3. 4. To study gene expression Novel promoter identification Disease diagnostics Gene regulation studies

Copy Number Analysis (useful for transgenic and disease research): In copy number analysis one can know the exact number of copies of a gene or transgene present in the genome of the sample. This is done by comparing the PCR reaction with the standard graph generated by the known copy number standards (Figure 3). Eco-RTPCR supports all the chemistries and assays used for knowing the copy number variation. It can diagnose copy number of 1 copy to 10000 copies with single copy number resolution.

The copy number analysis using RTPCR can be used for the following applications: 1. For Copy Number Variation (CNV) analysis of a particular gene or RNA across a population. 2. For determining the viral load or pathogen load. 3. For transgene copy number analysis in transgenic research (a substitute of Southernblotting and Northern-blotting based analysis) High Resolution Melt (HRM) in RTPCR: HRM is an added advantage of Eco-RTPCR, where the melt curve analysis is done with highest resolution of single base pair difference. This feature is best supported by Eco-RTPCR because the well to well difference is just 0.1 oC (other system have > 0.3 oC). HRM needs highest precision in melt curve analysis to disclose even the single base pair difference between two sequences. This is used for following applications: 1. Allelic Discrimination analysis 2. Mutation (even point mutations) detection 3. SNP genotyping, detection and gene-scanning: Eco-RTPCR can even be used for Class-3 and Class-4 SNPs (for Class-4 SNPs the melt curve shift is < 0.2 oC). 4. Diagnostics of mutations (deletions, duplications, inversions, point mutations, etc.) and chromosomal aberrations in genome and diseases related to them. 5. GC content in DNA library or a particular sequence. 6. In TILLING (Target Induced Local Lesions In Genome) experiments 7. DNA methylation studies (used in analysis of tumor, pseudotumor and normal tissues) Eco-RTPCR in Healthcare: Eco-RTPCR is best utilized in healthcare industry as a source for disease diagnostics. RTPCR based assays are approved by WHO for rapid diagnostics of various diseases like HIV, Bird-Flu, Swine-Flu, SARS coronavirus infection etc. It is also useful in diagnostics of many animal diseases like Foot and Mouth disease, Flu, etc. RTPCR provides rapid diagnostics of many viral diseases with highest sensitivity, accuracy and precision. Other than this the Eco-RTPCR is highly applicable in pre-natal disease diagnostics (Sickle Cell Anemia, Phenylketonuria, etc,), amniotic fluid RTPCR for mutation detection and early diagnostics of syndromes (Downs, Klinefelters, etc).

Eco-RTPCR can be also used in novel SNP detection, genotyping and population screening to understand and study the inheritance patterns of hereditary diseases and disorders (Retinitis Pigmentosa, Hemophilia, Cystic Fibrosis, PKU, Diabetes, etc.) Thus, Eco RTPCR can be of various applications in healthcare as well as research for which high sensitivity, accuracy and precision is required. It also provides rapid and highly sensitive diagnostics of diseases, mutations, syndromes, etc..