The BiosearchTech Blog

Molecular Beacons - Lights in the storm

Posted on Wed, Mar 10, 2010 @ 11:05 AM

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).

Read More

Topics: Real-time PCR, Referral from the Doctor

Dual-labeled BHQ probes - Performance to “dye” for

Posted on Wed, Feb 24, 2010 @ 10:20 AM

A "Referral from the Doctor" Blog Article-  

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

Structure
 

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
Read More

Topics: Real-time PCR, Referral from the Doctor

This Year's MVP (Most Valuable Practice): Touchdown PCR

Posted on Thu, Feb 04, 2010 @ 10:39 AM

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.

 

Read More

Topics: Real-time PCR, Referral from the Doctor

An Alternative to DNAse I heat inactivation: LiCl precipitation

Posted on Wed, Jan 13, 2010 @ 10:04 AM

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 75oC 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.

Read More

Topics: Real-time PCR, Referral from the Doctor

Slope Efficiency Equals Manageable and Corrected PCR

Posted on Wed, Dec 09, 2009 @ 12:01 PM

A "Referral from the Doctor" Blog Article-  

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

Read More

Topics: Real-time PCR, Referral from the Doctor

Bringing Back a Champion: Competitive Quantitative Real-time RT-PCR

Posted on Wed, Nov 18, 2009 @ 08:08 AM

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.

Read More

Topics: Real-time PCR, Referral from the Doctor

The New Favorite Reference Gene: 36B4, a.k.a. RPLP0

Posted on Wed, Oct 14, 2009 @ 08:06 AM

A "Referral from the Doctor" Blog Article-

Ribosomes are comprised of two subunits, one small (40S) and one large (60S). The 36B4 gene encodes an acidic ribosomal phosphoprotein P0 (RPLP0), which forms tight associations with the smaller 40S protein (L12). 36B4 protein is part of a pentameric complex that forms a stem-like structure, protruding into the cytoplasm, off of the 60S subunit. This protrusion functions to support the GTPase steps in the translocation of protein synthesis. Why is 36B4 fast becoming the new reference gene for probe-based real time RT-PCR?
Read More

Topics: Real-time PCR, Custom Oligos, Referral from the Doctor

What Genes are Hot, What Genes are Not

Posted on Wed, Oct 07, 2009 @ 02:41 PM

 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)

Read More

Topics: Real-time PCR, Referral from the Doctor

Subscribe to our blog

About Biosearch Technologies

As the inventor of the BHQ dyes, Biosearch Technologies synthesizes sophisticated oligos for real time qPCR, molecular diagnostics, and more!  Visit our home page to view our products and services.

Follow @BiosearchTech on Twitter

Become a Fan on Facebook!