Alkaline hydrolysis to remove potentially infectious viral RNA contaminants from DNA

Table 2 Optimization of RNA hydrolysis while maintaining viral and plasmid DNA integrity

Sample ID	Heat (min)	DNA	SCR RNA	NaOH	HCl	TE	SCR PCR	ASF PCR	CSF PCR	DNA integrity
1- ASF DNA control	0	35 μl	7 μl	0 μl	0 μl	30 μl	+	+	NA	+
2- CSF DNA control	0	35 μl	7 μl	0 μl	0 μl	30 μl	+	NA	+	+
3- Plasmid DNA control	0	35 μl	7 μl	0 μl	0 μl	30 μl	+	NA	NA	+
4- ASF DNA heated	30	35 μl	7 μl	0 μl	0 μl	30 μl	+	+	NA	+
5- CSF DNA heated	30	35 μl	7 μl	0 μl	0 μl	30 μl	+	NA	+	+
6- Plasmid DNA heated	30	35 μl	7 μl	0 μl	0 μl	30 μl	+	NA	NA	+
7- ASF DNA treated	30	35 μl	7 μl	15 μl	15 μl	0 μl	-	+	NA	+
8- CSF DNA treated	30	35 μl	7 μl	15 μl	15 μl	0 μl	+	NA	+	+
9- Plasmid DNA treated	30	35 μl	7 μl	15 μl	15 μl	0 μl	+	NA	NA	+^a
10- ASF DNA treated	45	35 μl	7 μl	15 μl	15 μl	0 μl	-	Inc^b	NA	+
11- CSF DNA treated	45	35 μl	7 μl	15 μl	15 μl	0 μl	+	NA	+	+
12- Plasmid DNA treated	45	35 μl	7 μl	15 μl	15 μl	0 μl	-	NA	NA	+^a
13- ASF DNA treated	60	35 μl	7 μl	15 μl	15 μl	0 μl	-	Inc^b	NA	+
14- CSF DNA treated	60	35 μl	7 μl	15 μl	15 μl	0 μl	-	NA	+	+
15- Plasmid DNA treated	60	35 μl	7 μl	15 μl	15 μl	0 μl	-	NA	NA	+^a
16- SCR in TE	0	0 μl	7 μl	0 μl	0 μl	65 μl	+	NA	NA	NA
17- SCR in salt	0	0 μl	7 μl	15μl^c	15μl^c	35 μl	+	NA	NA	NA
18- SCR undilute control	0	0 μl	2.5 μl	0 μl	0 μl	0 μl	+	NA	NA	NA

Samples containing various combinations of ASF DNA, CSF cDNA, and plasmid DNA, under various treatment conditions. All samples contained SCR, and results for verification of SCR degradation and maintenance of plasmid DNA integrity are listed. NaOH and heat treatment incubation time used in this study range from 0 to 60 min. Following heat treatment for various times at alkaline pH, all samples were boiled for 10 min at neutral pH. ^aSlight nicking or denatured ssDNA was observed as compared to untreated control plasmid. ^bInconclusive rPCR result (Inc) due to high concentration of DNA template. ^cNeutralized salt solution (equal parts 1 N NaOH and 1 N HCl) reagent used as a positive control. NA indicates not analyzed

ISSN: 1743-422X