eoe sub articles

EoE Sub Articles

1. Quality Control of Genomic DNA
NOTE: Genomic DNA (gDNA) was extracted from peripheral blood using the well-established silica-membrane-based DNA purification method. In advance of the below procedures, the concentration of gDNA was determined using a spectrophotometer.
<ol>
	<li>Prepare the gDNA sample at a concentration of 10-100 ng/&#181;L.</li>
	<li>Add 10 &#181;L of DNA sample buffer to an 8-tube strip.</li>
	<li>Add 1 &#181;L of DNA ladder or&#160;gDNA to the tubes. ...

chip-in-a-tube-based digital pcr for quantification of single nucleotide variants

Source: Kahyo, T., et al. <a href="https://app.jove.com/v/58199">Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format.</a> J. Vis. Exp. (2018).
This video demonstrates the utility of digital PCR in a chip-in-a-tube format for the detection of single nucleotide variations in DNA samples. A single PCR reaction is partitioned into chambers that act as independent PCR reactions, and the detection of fluorescence signals from the amplified targets in the chambers is used to compute the frequency of the variant allele in the sample.

<img alt="Figure 1" src="/files/ftp_upload/21362/21362_Figure1.jpg" /> 
Figure 1: Points to note for the dPCR assay with chip-in-a-tube format.&#160;(A) This panel shows how to set up the 8-tube strip in the autoloader. (B) This panel shows broken chips. (C) This panel shows how the chip surfaces just after (i) loading and (ii) sealing. (iii) A puddle of liquid is visible in the case of an incomplete sealing. (<stron...

Chip-in-a-Tube-Based Digital PCR for Quantification of Single Nucleotide Variants

Source: Kahyo, T., et al. Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the ...

Digital polymerase chain reaction &#8212; or dPCR &#8212; is useful for quantifying single nucleotide variants &#8212; or SNVs &#8212; a mutation where the substitution of a single nucleotide at a specific position in the genome creates two nucleotide variations or alleles.
To begin quantification using the chip-in-a-tube dPCR technique, take a DNA sample containing SNVs &#8212; the mutant allele, and the corresponding wild-type allele. Add a mixture containing a thermostable DNA polymerase enzyme, dNTPs, and primers.
Next, add allele-specific oligonucleotide probes &#8212; labeled with reporters of different-colored fluorescence to distinguish between the alleles &#8212; and a quencher molecule. The proximity of the quencher to the reporter suppresses its fluorescence.
Partition the solution into the reaction chambers of a microfluidic chip. Each chamber containing an allele serves as an independent reaction vessel.
Place the tube with the chip inside a thermal cycler, and start the reaction. At a high temperature, the double-stranded DNA denatures into single strands. Lower the temperature to anneal the primers and oligonucleotide probes to their complementary regions.
At an appropriate extension temperature, the DNA polymerase extends the primers and cleaves the probe. Distance from the quencher enables the reporter&#39;s fluorescence emission.
Read the different-colored fluorescence signals, and create a position plot to detect the chambers containing the alleles.
To determine the frequency of the mutant allele &#8212; the variant allele fraction &#8212; compute the ratio of chambers containing the mutant versus the wild-type allele.

Chip-in-a-Tube-Based Digital PCR for Quantification of Single Nucleotide Variants

Overview