Biomedical Engineering Reference
In-Depth Information
Fig. 6. Stencil-aided mutation analysis (SAMA) of mutant K
-RAS
DNA in the pres-
ence of different amounts of wild-type allele. One nanogram of DNA from SW480 cell
line carrying two mutated alleles at codon 12 of the K
-RAS
gene was mixed with differ-
ent amounts of DNA from peripheral lymphocytes of healthy donors and subjected to
SAMA following by enriched polymerase chain reaction (157-bp amplicon).
Fig. 7. Detection of K
-RAS
mutation in Tr-DNA from three patients with pancreatic
cancer.
Lanes 1-3, enriched polymerase chain reaction (PCR); lanes 4-6, stencil-aided
mutation analysis (SAMA) plus enriched PCR.
4. Notes
1.
We have not observed a significant difference between DNA isolated from urine
specimens obtained at different time points during the day. However, we prefer
not to use the first morning urine specimens, because additional DNA degrada-
tion may have occurred during the hours of sleep.
2.
It is highly desirable to inhibit nucleases that have been found in some urine
samples. EDTA is added to inhibit DNase-I type nucleases, and a relatively high
pH inhibits acidic nucleases.
3.
If there is not the specific purpose of analyzing cell free DNA, we would normally
isolate DNA from unfractionated urine specimens because a portion of cell free
DNA has a tendency to co-sediment with cells during centrifugation. Although
this effect cannot be entirely prevented, it can be minimized by centrifugation at
room temperature and washing the sediment.
4.
To avoid a very significant loss of Tr-DNA, commercial kits designed for isola-
tion of high molecular weight genomic DNA should not be used for Tr-DNA
purification.
5.
Figure 3
illustrates the advantages of a shorter amplicon size when PCR is
performed with small DNA templates. Most of the long amplicons are out of
