Research Update
CleanAmp™ dNTPs Improve 3C-qPCR
Spatial conformations of chromosomes, such as loops and interchromosomal connections, play important roles in gene expression, replication, DNA repair and other fundamental cellular processes1. A pioneering and major methodological advance for investigation of higher-order chromosome structure, called Chromosome Conformation Capture (3C), was reported in 2002 by Dekker et al.2 and has been cited in more than 200 publications. The workflow for 3C involves formaldehyde fixation of intact nuclei to crosslink proteins to other proteins and DNA, restriction digestion, intramolecular ligation of DNA, reversal of crosslinking and then PCR-based analysis of the frequency with which pairs of selected DNA fragments are crosslinked. Accurate measurements of crosslinking frequencies require extremely accurate quantification techniques. Real-time probe-based qPCR for 3C assays, which is called 3C-qPCR, has been reported3 as being able to more accurately determine crosslinking frequencies compared to semiquantitative 3C strategies that rely on measuring the intensity of ethidium bromide-stained PCR products separated by gel electrophoresis. The original 3C-qPCR protocol3 was recently modified4 by Forné et al. to allow real-time detection using an intercalator dye instead of probes, based in part upon replacement of standard dNTPs with TriLink’s CleanAmp™ dNTPs. This newly improved 3C-qPCR procedure was used to elucidate fundamental aspects of chromatin dynamics in mammals, thus contributing to a better understanding of genome organization within chromosomal territories4. In another report5, the CleanAmp™ dNTP-enabled 3C-qPCR method was used to investigate a novel imprinted domain that led to a novel model for chromatin folding on the paternal chromosome.
Learn More About CleanAmp™ dNTPs
Order CleanAmp™ Products
View Latest CleanAmp™ Posters |
|
|
Ask An Expert Featured Question
Q. Do CleanAmp™ dNTPs form a modified amplicon?
A. No, CleanAmp™ dNTPs are reverted back to the corresponding standard dNTP prior to being incorporated into the amplicon. The 3’-hydroxyl modification blocks enzyme incorporation until it is removed during heat activation. After heat activation, the enzyme efficiently incorporates the corresponding standard dNTP.
Learn More I Ask An Expert |
|
