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. Maslinic acid suppressed NF-kB (p65) binding activity in a time-dependent manner, with 8.3%, 42.9%, 63% and 70% inhibition observed at 1,2,4, and 8 h treatment (Fig.
After purification competitor DNA
, target DNA (fungi medium culture) and serial dilution of competitor DNA
([10.sup.-3],[10.sup.-4], [10.sup.-5], [10.sup.-6] and [10.sup.-7]) used in the QC-PCR and was shown to amplify under the same reaction condition and the same amplification efficiency as the target 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 0.1 pmol and 50 pg, respectively.
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.
Wild-type phagemid DNAs consisting of [10.sup.2], [10.sup.3], [10.sup.4], [10.sup.5], [10.sup.6], [10.sup.7], and [10.sup.8] copies/5 [micro]L were prepared and measured by C-PCR using the corresponding competitor DNA as described above.
Exonuclease III-generated series of homologous competitor DNA fragments for competitive PCR.
One explanation may be variations in the amounts of genomic DNA and/or competitor DNA
in the reaction.
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.
To overcome tube-to-tube, sample-to-sample, and even day-to-day amplification variations during PCR [5, 6], competitive PCR has been widely used, i.e., coamplification of a specific target DNA and known amounts of a competitor DNA