The BiosearchTech Blog

The largest recorded Ebola Virus Disease outbreak in history continues to devastate the West African region. This epidemic has presented a public health emergency to national and international disease control centers with aid workers tirelessly working to assist affected communities. Ebola is a single-stranded RNA virus that has five known strains, four of which are pathogenic to humans. The current outbreak is a result of the spread of the Zaire ebolavirus (EBOV) strain which previously averaged a 78% mortality rate ¹. In a recent update from the World Health Organization, upwards of 2,600 deaths have been reported as result of the viral infection over the last several months ².

Don’t waste time performing an extra step. Cut back on pipetting and order a ValuMix™ Assay to have your probes and primers delivered in one tube. The oligos are formulated according to the quantities you specify and delivered dry for maximal stability. ValuMix Assays expedites your research because they:

Do you remember when the most popular method of DNA amplification involved multiple hybridizations, followed by single or 2-primer extension and ligation steps? DNA amplification was a long and tedious process, and if people followed the saying “if it ain’t broke, don’t fix it” the world would certainly be in a different place today. Without rapid PCR assays, it could take several months for medical test results, innocent people would continue to overcrowd prisons, and less research will be achieved but more interns hired (someone needs to perform all those hybridizations).

A "Referral from the Doctor" Blog Article-

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive and
robust method for analyzing RNA. It has been used to corroborate the findings of other genetic methods, such as microarray and RNAse protection assays, and has become the gold standard for quantifying changes in gene expression. Stellaris FISH is a new RNA detection method that provides direct quantification of transcripts in situ, with potential to become the standard for mRNA and lncRNA interrogation using microscopy.

Non-squamous, Non-small cell lung cancer (NSCLC), the most common cause of death in the US and globally, has recently been dealt a significant blow as a result of collaborations among teams from UCSF Thoracic Oncology, Kaiser Permanente Research, and the China Clinical Trials Consortium (CCTC) uniting hospitals and universities across mainland China.  

A "Referral from the Doctor" Blog Article-

In qPCR, fluorescent modified oligos may be either linear or non-linear in structure. In either conformation, Förster resonance energy transfer (FRET) combined with the static quenching mechanism inhibits reporter dye emission until hybridization with the target (Read Quenching Mechanisms in Probes to learn more about FRET and static quenching). Upon hybridization the quencher and reporter dye are separated spatially, interrupting the quenching mechanisms and permitting fluorescence emission from the reporter dye. The increase in signal intensity with repeated PCR cycles indicates the accumulation of product and allows for accurate quantification of template. Gene expression analysis by qPCR is dependent upon direct comparisons between the normalized fluorescence emission of individual samples against a standard curve or through ‘relative’ comparison against expression of an internal calibrator gene.

How do I know how bright the dye will be?

A "Referral from the Doctor" Blog Article-

Fluorescent probes are used in biochemical assays to monitor specific events such as binding, cleavage or conformational changes. Dual-labeled probes with a fluorophore and a quencher have many applications in genetic analysis. The efficiency of fluorescence quenching is very distance dependent – if the reporter fluorophore and quencher are far apart, there is fluorescence; if the reporter and quencher are close together in space, fluorescence is suppressed. Typically, the reporter and quencher are placed at specific sites in an oligonucleotide such that a change in their distance will produce a maximal change in fluorescence and effectively signal the event being monitored (often hybridization or nuclease activity). The oligonucleotide acts as a flexible tether linking the fluorescent reporter and quencher. Below, we present fluorescence quenching mechanisms for dual-labeled oligonucleotides in genetic analysis.

A "Referral from the Doctor" Blog Article-

Quantitative real-time PCR (qPCR) was developed as a means to detect the presence of target nucleic acids and is routinely used to quantify gene expression through reverse transcription of RNA transcripts.  The detection method depends on a signaling molecule, often a dual-labeled oligonucleotide with a fluorophore (reporter) dye at the 5’ end and quencher dye at the 3’ end. The development of new reporter dyes, true dark quenchers and advancements in probe sophistication enable researchers to improve their genetic analysis.  One significant improvement is in the application of multiplexed qPCR to yield additional data and confidence in the results.

A "Referral from the Doctor" Blog Article-

Quantitative real-time PCR (qPCR) is based upon the fractional cycle number at which a replicating sample of target DNA accumulates sufficient fluorescence to cross an arbitrary threshold. The threshold is either manually selected or auto-selected to fall several standard deviations above baseline fluorescence and below the plateau phase, where the amplification begins to attenuate. Typically, the threshold is adjusted to the mid-point of the exponential phase of the PCR, at a location suitable for all samples in the experiment. The C_T (cycles to threshold) value for a given reaction is defined as the cycle number at which the fluorescence emission intersects the fixed threshold.

Biosearch Technologies, Inc. (Biosearch), a leading supplier of sophisticated oligonucleotide components to the rapidly growing molecular diagnostics industry, and IMDx (privately held), a developer and manufacturer of innovative, clinically impactful molecular test solutions, today announced that Biosearch has licensed access to the BHQ®, CAL Fluor® and Quasar® patents to IMDx.  In addition, Biosearch will manufacture cGMP oligonucleotides for IMDx for use in human in-vitro diagnostics.

A "Referral from the Doctor" Blog Article-

ROX in qPCR

Quantitative real-time RT-PCR was developed as a means of quantifying gene expression either relatively or absolutely, with accurate and reproducible results. Despite advances in dye chemistries, variability continues to haunt research scientists.  One effort to reduce the observed variability between technical replicates was to include the ROX dye (5- or 6-carboxy-X-rhodamine) as a passive reference in the PCR master mix.

Biosearch Technologies, Inc. (Biosearch), a leading supplier of sophisticated oligonucleotide components to the rapidly growing molecular diagnostics industry, today announced that the company has licensed access to the BHQ®, CAL Fluor® and Quasar® patents to DuPont Qualicon.

Biosearch Technologies, Inc. (Biosearch), a leading supplier of sophisticated oligonucleotide components to the rapidly growing molecular diagnostics industry, today announced that the company has licensed access to the BHQ®, CAL Fluor® and Quasar® patents to Seegene (KOSDAQ; Korea Securities Dealers Automated Quotations).

Biosearch Technologies today announces that it has entered into a new license relationship with QIAGEN (NASDAQ: QIA, Frankfurt Prime Standard) providing broad commercialisation rights to Scorpions Primers.  This agreement allows Biosearch the right to manufacture, catalog, and sell Scorpions primer assays into the research, applied, and infectious disease testing markets.

A "Referral from the Doctor" Blog Article-

When the polymerase chain reaction (PCR) was first described, the Klenow fragment derived from the Escherichia coli DNA Polymerase I was the paramount enzyme for sequence extension. Due to its lack of stability at high temperature, it needs be replenished before each cycle. Upon the discovery of thermophilic bacteria which thrive at temperatures greater than 45 °C, heat-stable polymerases which function at higher temperatures were investigated in an effort to eliminate the need to replenish enzyme following each denaturation cycle.

A "Referral from the Doctor" Blog Article-

Research trends in laboratories today increasingly steer towards gene expression analysis and genetic testing, often in the form of qPCR. As reproducibility is essential to genetic research it is imperative that scientists know the fundamentals of micro-volume pipetting.

Forward and Reverse Pipetting: This discussion is limited to the use of manual pipettors. Electronic pipettors are capable of other pipetting techniques such as dispensing, sequential dispensing and diluting which are not discussed here.

Buy one CAL Fluor® or Quasar® -BHQ® Probe and get a FREE ValuProbe^TM BHQ Probe* and calibration standard ($190 in savings!). Simply enter promo code CFQVP upon checkout to receive this offer. Take advantage of this promotion before it expires on June 30, 2010.

Biosearch Technologies offers CAL Fluor and Quasar dyes that span the spectrum, for 2-plex, 3-plex, 4-plex, 5-plex, and even 6-plex PCR. They perfectly complement the Black Hole Quencher dyes, a true dark quencher that extinguishes signal from any fluorophore.

We are pleased to introduce ValuPanel Reagents, a new product line of mission-critical probe and primer sets that discriminate different strains of pathogens. This product line culminated from our successful collaboration with Centers for Disease Control and Prevention* (CDC) in response to the 2009 H1N1 pandemic. As of now, ValuPanel Reagents are available for both 2009 H1N1 and seasonal Influenza A subtyping.

A "Referral from the Doctor" Blog Article-  

Scorpions®Primers are dual-labeled FRET probes that combine a Molecular Beacon®-like probe structure and a PCR primer element in a single oligonucleotide, allowing for specific target amplification and advanced target detection through a unimolecular or single oligonucleotide driven mechanism.

Q. What parts are similar to dual-labeled Black Hole Quencher® (BHQ) probes?
A BHQ dye and a 5'-fluorophore covalently bound to the oligonucleotide termini.

A "Referral from the Doctor" Blog Article-

Molecular Beacons are a special type of dual-labeled oligonucleotide probe. Beacons are hairpin loop structures with a 5'-fluorophore and a 3'-quencher dye. The stem region is a short sequence of 5-7 complementary bases. The loop sequence is complementary to the target sequence. In the absence of target or prior to amplification, the stem anneals to form a closed hairpin conformation which holds the reporter and quencher close together to enable efficient FRET quenching and to promote contact or static quenching. The beacon is engineered such that the probe-target hybrid is more stable than the closed hairpin conformation, which is respectively more stable than a probe-mismatch hybrid. Therefore it is only in the event of a perfect-match hybridization that signal occurs, allowing beacons to discriminate mismatches as small as a single nucleotide polymorphism (SNP).

A BHQ® probe is a dual-labeled oligonucleotide covalently labeled with a fluorophore and a Black Hole Quencher® (BHQ) dye.

Q. What are the main components of a dual-labeled Black Hole Quencher® (BHQ) probe?

• An oligonucleotide, typically 30 bases long
• A 3' BHQ dye
• A 5'-fluorophore (reporter) dye

A "Referral from the Doctor" Blog Article-  

The aim of the Polymerase Chain Reaction (PCR) method is to amplify or increase the number of copies of a target sequence. Most often that sequence is part of a gene of interest and the total copy number or relative copy number of that transcript will impart some knowledge to the researcher regarding the impact of a drug, disease status of a sample or may represent a control condition. In some situations, the copy numbers of a particular transcript are very low and it may be necessary to nudge the PCR reaction in favor of amplifying the exact splice variant, family member or a construct the researcher wishes to examine.

A "Referral from the Doctor" Blog Article-

Genomic DNA contamination has been a hot topic for qPCR researchers since the beginning. It is impossible to remove all DNA from RNA preparations without additional steps. The most commonly used technique is to incubate with DNAse I. The deoxyribonuclease I (DNAse I) enzyme catalyzes the hydrolytic cleavage of phosphodiester linkages adjacent to a pyrimidine nucleotide. It acts on single-stranded DNA, double-stranded DNA, and chromatin. DNAse I activity will degrade trace amounts of genomic DNA (up to 10 µg/mL) that could otherwise result in falsely positive signals in subsequent RT-PCR reactions. DNAse I is a heat-inactivated nuclease, requiring both the presence of EDTA and temperatures of 75^oC for 5 minutes for complete inactivation. The extreme temperatures associated with heat-inactivation of the enzyme may cause damage to the RNA through chemical mediated degradation if even small amounts of metal ions are present. Lower temperatures will not fully inactivate the DNAse before reverse transcription of the RNA to cDNA. Because we cannot discriminate between cDNA and DNA amplification products, initial copy number determination is compromised.

1. Instrumentation and Dye Selection
This one is pretty easy. For a successful multiplex, your instrument needs to be able to excite each of your reporter dyes. Although there are dyes on the market that excite in the far red, you'll be safe using an instrument which excites over the visible spectrum (350-750nm). Biosearch Technologies has developed two essential tools to assist you with dye selection:

A "Referral from the Doctor" Blog Article-  

ε = m.c.2 (shall I explain? slope efficiency equals manageable and corrected PCR)

Biosearch Technologies recently launched a video, Making of the Swine Flu Test Kit - Inside Biosearch Technologies, which had debuted last week at qPCR Symposium 2009 held in Millbrae, California.  This video is an accurate re-enactment of Biosearch Technologies' rapid production of the H1N1 (Swine Flu) test kit that was developed by the Centers for Disease Control and Prevention (CDC).

A "Referral from the Doctor" Blog Article-

Competitive qRT-PCR has become a lost art. Early titans of probe-based PCR used this technique to minimize variability between samples and experiments due to differences in reverse transcription and amplification efficiency. This technique facilitates the absolute measurement of initial gene target copy number based on known copy numbers of a control template.

This is accomplished through the addition of an internal control, a competitor sequence to the mRNA target in the sample. The competitor sequence is similar enough to use the same primer pairs as the target sequence but has a unique central sequence allowing for analysis with a different probe and reporter. Plasmid DNA or in-vitro transcribed RNA is commonly used as a competitor sequence and standard for quantification. To detect the initial copy number of a gene of interest, multiple replicates of a fixed amount of the target mRNA are dispensed into tubes. In the same tubes, 10 fold serial dilutions of competitor RNA are "spiked" in sequentially. Using one set of primers and two fluorogenic probes labeled with different dyes, real-time PCR data can be collected for both sequences simultaneously.

Biosearch Technologies announced today that the company has licensed from the CDC the novel H1N1 Influenza signatures along with the Influenza A sub-typing panel signatures.  This licensing agreement with the CDC makes Biosearch Technologies the first oligo manufacturer to have the right to manufacture and sell dual-labeled probes and primers bearing the H1N1 and Influenza A sub-typing signatures.

Micronics, Inc., a leading developer of point-of-care molecular diagnostic products, announced today that it has entered into a license agreement with Biosearch Technologies, Inc. for the right to make, have made, use and sell Micronics' products that incorporate Biosearch's proprietary fluorophores and quencher dyes for nucleic acid assays. Under the terms of the worldwide license, Biosearch has granted Micronics a royalty-free use of certain of its chemistries for inclusion in Micronics' infectious disease tests for sale in underdeveloped countries.

A "Referral from the Doctor" Blog Article-

Biosearch Technologies has been selected for two Small Business Innovative Research (SBIR) grants from the Department of Defense (DoD). Both Phase 1 grants calls for the development of highly sensitive analyte specific reagents (ASRs) for a total of twelve pathogens.  The ASRs are qPCR based, but modified for field deployment.  Both grants include the following target pathogens: Dengue, Rift Valley Fever, Sand Fly Fever, Crimean-Congo Hemorrhagic Fever, Tick-Borne Encephalitis, Chikungunya, causative agents of typhus, spotted fever, anaplasmosis, Ehrlichioses, and Q fever.  If Phase I turns out to be a success, Biosearch expects this project to lead into a multiyear Phase II award.

 A "Referral from the Doctor" Blog Article-

 Top 10 Reference Genes for probe-based Real-time PCR in 2009:

• Acidic Ribosomal Phosphoprotein P0 (RPLP0 or 36B4)
• ATP-synthase subunit 5B (A5B)
• Tumor Protein Translationally controlled 1 (TPT1)
• Signal Recognition Particle 14kDa (SRP14)
• TATA-Binding Protein (TBP)
• Eukaryotic Elongation Factor 1A1 (EEF1A1)
• Hypoxanthine Phosphoribosyl-Transferase 1 (HPRT1)
• poly-Ubiquitin (Ubi)

When to use an internal Black Hole Quencher (BHQ) label in a fluorescence-quenched probe for real-time qPCR.

When the sequence length of your probe is 30 bases or shorter, we recommend selecting a conventional BHQ label for the 3' end of your probe. We have noticed, however, that probe lengths longer than 30 bases exhibit high background noise and are frequently nonfunctional. They are too long for efficient FRET quenching, a mechanism that is highly dependent upon the distance between the fluorophore and quencher. A shorter average distance between fluorophore and quencher is optimal for a FRET quenching environment, which is common in real-time qPCR experiments.

We have done extensive research to show that the difference in purity specifications does not compromise the quality or performance in assays using the salt-free primer pairs instead of the cartridge purified primer pairs. You can review the Biosearch-conducted purification study by downloading this report.

Since its introduction in 2005, the ValuProbe BHQ Probe has become Biosearch Technologies' most popular product sold to the real-time qPCR industry.  Many molecular biologists choose the ValuProbe BHQ Probe because they are quality, superior performing fluorescence-quenched probes that work great for validating and optimizing real-time qPCR assays.