PREMIER Biosoft

SYBR Green Assay Chemistry - Primer Design Considerations.

PREMIER Biosoft — Fri, 26 Dec 2008 06:16:00 GMT

Polymerase chain reaction (PCR) is construed as one of the pioneering molecular biology techniques of the 90's. In fact, it was THE technique that completely revolutionized molecular biology. Its been more than a decade of PCR domination in molecular biology and clinical diagnostic labs, worldwide. The technique has evolved during all these years, and now, it is real time PCR (qPCR) which is getting the same attention and is becoming one of the most widely used molecular biology techniques of this new millennium.

Real Time PCR quantifies the amount of nucleic acid in a given sample and its applications include gene quantification, pathogen detection and validation of drug targets. Quantitation is not a new idea in molecular biology. HIV viral loads in AIDS patients have been analyzed since decades now. These days, the focus is on quantitation because of the advancements in genomics research and the emergence of real time quantitative PCR.

Let us, in this series of blog posts, have a look at the different chemistries that have been developed over all these years. These chemistries have their own advantages and disadvantages which will be discussed in some other series. Let us just look at the technical aspects and major players involved in them -

SYBR Green Primer Design Guidelines

1) The length of the product i.e. the amplicon should be 250 - 400 bp.

2) During the SYBR green primers design, template regions with secondary structures should be avoided. The template may form loops and hairpins in the regions where a lot of complementary bases exist. This hinders the primers from annealing and extension. Thus it results in preventing the product formation.

3) The melting temperature i.e. the Tm should be 60 - 65 oC.

4) Primers should be checked for dimers, both self and cross. Dimers should be avoided as they greatly reduce the availability of primer for reaction. Reference paper for elimination of primer dimer artifacts (http://www.gene-quantification.de/vandesompele-papae-2002.pdf).

5) G/C content should be 20-80% in the regions where the primers are being designed. The bonding between G and C is strong, thus it requires more energy to break which may not denature well during PCR cycling. This results in the less efficient PCR reaction. So the primers with long runs of a G should generally be avoided as it can cause breathing of primer and also aid mispriming.

TroubleShooting guide (http://www.abgene.com/downloads/Guide_QPCR-sybr.pdf)

NetPrimer - Free Oligo/Primer Properties Calculator

A Free Domain for Molecular Biologists

PREMIER Biosoft — Thu, 04 Sep 2008 05:59:00 GMT

Molecular Biology techniques need tools, both software and hardware for successful accomplishment. Here is a free domain that hosts some common tools by PREMIER Biosoft for PCR, qPCR and mutagenesis using Stratagene's QuikChange kit

Free Molecular Biology Tools.

Technical qPCR guide

PREMIER Biosoft — Wed, 28 May 2008 04:45:00 GMT

A great reference for the Bio-IT community. We have all been doing white paper marketing and marketing via article publishing. A step in here is this qPCR technical guide. qPCR or real time PCR is one of the most commonly used molecular technique for identification of a target gene. The technique is accurate, sensitive and less time consuming. The dependability of results is high as compared to hybridization based techniques such as northern blotting etc.

The technical guide on qPCR contains each and every detail related to the qPCR experimentation starting from a technology introduction to primer and probe design, instrumentation, application guide, optimization and troubleshooting sections.

Check it out on the Sigma-Aldrich website at:
http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Molecular_Biology/PCR/Product_Lines/Quantitative_PCR/qPCR_Technical_Guide.html

PREMIER Biosoft enters the new arena of Tissue Microarrays

PREMIER Biosoft — Thu, 03 Apr 2008 05:22:00 GMT

With the release of its TMA Foresight software, PREMIER Biosoft has entered the increasingly popular Tissue Microarray field.

TMA Foresight is a concept transformed into a software by PREMIER. The software is a statistical package for IHC (Immunohistochemistry) research. The classical approach of recruiting bio-statisticians is now fading. TMA Foresight works as a bio-statistician for clinicians, pathologists and researchers for analyzing TMA data.

The software supports statistical techniques such as Kaplan Meier plots, Cox Regression, Hierarchical Clustering, Principal Component Analysis and Correlation analysis. It outputs results in plain English about the relatedness of biomarkers and clustering of patients.

TMA Foresight link

A new, free real time PCR oligo calculator

PREMIER Biosoft — Mon, 31 Mar 2008 04:42:00 GMT

PREMIER Biosoft International; the leading provider of software solutions for molecular and cell biology research scientists, released a free online tool for analysis of primers and probes used in real time qPCR assays.

The Beacon Designer free edition tool is a unique solution for the research community working with the advanced version of the PCR chemistry; qPCR. This web-based qPCR design tool predicts the important properties of the SYBR® Green primers and TaqMan® primer/probe sets using the latest thermodynamic algorithms. It analyzes primers and probes for all important undesired interactions including hairpins, dimers, runs and repeats and calculates the Tm, GC% and length.

The full version of the software is a standalone product of designing real time PCR assays and includes design support for all the popular real time PCR chemistries and is used in the laboratories around the world.

With the release of this tool, Premier Biosoft has added to its existing offering of free tools that research scientists working in the molecular and cell biology field can use to accelerate their research. Premier Biosoft already has an oligo properties calculator called NetPrimer and a desktop tool to design primers for Stratagene's QuikChange® site directed mutagenesis kits, called MutaPrimer.

"Beacon Designer free edition is a token of our appreciation towards the scientific community and the researchers who have long supported our products and have made them a part of their research." says Kay Brown, V.P. Business Development and Marketing.

Direct link to the free qPCR design tool.

Press Release on Premier Biosoft website.

About PREMIER Biosoft International:
Founded in 1994, the company is a group of computer scientists and molecular biologists dedicated to producing cutting edge intuitive software for the molecular biologist. The goal of the company is to study the most recent innovations in molecular biology and translate them into software products that aid biologists. Additional information about PREMIER Biosoft International can be found at http://www.PremierBiosoft.com