These include reactions performed in the absence of DNA-binding protein extract (blank), reactions with non-specific competitor DNA and reactions with specific competitor DNA.
7A, the NF-kB (p65) binding activity in Raji cells was specific; presence of non-specific competitor (mutant DNA) did not affect the NF-kB binding activity in Raji cells but was significantly reduced in the presence of specific competitor DNA.
After purification competitor DNA
, target DNA (fungi medium culture) and serial dilution of competitor DNA
Consequently we chose 3000 copies of single-stranded competitor DNA
for the estimation of total DNA and 300 copies of competitor DNA
for the estimation of fetal DNA.
For the nonspecific competitor DNA assays, everything was identical except labeled F-DNA and Ape1 were present at 0.
Briefly, after incubation of Ape1 protein with an inhibitory metal, equimolar or 10-fold excess nonspecific competitor DNA (relative to the labeled F-DNA) was added simultaneously with radiolabeled abasic DNA substrate, and AP site incision was then measured.
8] copies/5 [micro]L were prepared and measured by C-PCR using the corresponding competitor DNA
as described above.
The peak areas of target to known copy numbers of competitor DNA
allowed quantification of the target without any assumption about PCR efficiency of different targets and samples.
For coamplification of both target and competitor DNA
, a B19 parvovirus DNA internal sequence of 184 by corresponding to nt 1652-1835 was chosen.
The effect of competitor DNA
on the thin film assay was analyzed by addition of a 5000-fold excess of salmon sperm DNA to the target.
coamplification of a specific target DNA and known amounts of a competitor DNA