Biomedical Engineering Reference
In-Depth Information
This finding implies that circulating EBV DNA in NPC patients mainly con-
sists of short DNA fragments of less than 181 bp and that less than 1% of the
plasma EBV DNA molecules are longer than 500 bp.
4. Notes
1.
The described plasma processing protocol has been shown to effectively remove
blood cells from plasma. Incomplete removal of blood cells would lead to an
apparent increase in the size of circulating DNA.
2.
The size of circulating DNA would be affected by sample types and preanalytical
factors including the delay in separation of cellular components and repeated
freezing and thawing of plasma samples. Blood clotting and a greater than 6-h
delay in the separation of plasma from cellular components would lead to the
release of high-molecular-weight DNA from blood cells into the fluid compart-
ment ( 14 , 15 ) , and thus would result in an apparent lengthening of circulating
genomic DNA in size analysis. On the other hand, repeated freezing and thawing
of plasma samples would result in increased fragmentation of circulating DNA
( 15 ) . In contrast, no significant fragmentation of DNA would be observed after
repeated freezing and thawing of extracted DNA for up to three times ( 15 ) .
3.
During the optimization of assays, we also evaluated the differences between the
use of a standard TaqMan probe and an MGB probe and found that the assays
using MGB probe had lower detection limits when the amplicon lengths went
beyond 400 bp. This may be due to the better binding of the MGB probe to the
DNA template during the new strand synthesis.
4.
Namalwa cell line is a diploid cell line containing two integrated EBV genomes/
cell and it is incapable of producing free EBV. A conversion factor of 6.6 pg of
DNA per diploid cell was used for copy number conversion. As the DNA mol-
ecules extracted from the Namalwa cell line are of high molecular weight, they
could be detected by assays with different amplicon lengths.
5.
All reactions should be run in duplicates and the average of two threshold cycle
(C T ) values is used for the analysis for each sample of unknown quantity and for
each calibration sample. Multiple water blanks are also required as negative con-
trols and extra volume for compensation for pipetting errors (200
L) is required
when preparing the master mix. Therefore, the number of reactions N is equal to
2
µ
×
(no. of unknown samples + 14).
6.
The addition of UNG into the reaction mixture and the use of dUTP instead of
dTTP is an anti-contamination strategy ( 16 ) . As dUTP is used in all PCR reac-
tions carried out in our laboratory, all carryover PCR products would be destroyed
by enzymatic digestion by the UNG during the 2-min incubation at 50°C ( step 1
of the thermal profile). In step 2 of the thermal profile, the UNG is degraded and
the AmpliTaq Gold polymerase is activated.
7.
For circulating DNA size analysis, a separate calibration curve (PCR amplifica-
tion) should be set up for each real-time PCR of different amplicon sizes. This
size-specific calibration curve can minimize the effect of the different efficien-
cies of PCRs.
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