Adaptation of a chemiluminescent DNA detection system to strongly positively charged nylon membranes: single copy gene detection in human genome.
Nonradioactive methods for detection of genes by Southern hybridization are limited to neutral or positively charged membranes which are supplied or explicitly recommended by the manufacturers of the chemiluminescent detection systems. The results of such kits with a known strongly positively charged nylon membrane (Genescreen plus) are disappointing. The adaption of a chemiluminescent detection system to such a strongly positively charged nylon membrane is demonstrated in this communication. The modifications include a blocking step (1% SDS) before prehybridization, use of 5% SDS during hybridization, and 5% SDS and 1% BSA during detection. The increased sensitivity of such a highly positively charged nylon membrane enabled us to detect 2.5 pg of target DNA. The detection of both the alleles of a single copy polymorphic target (YNZ22) in the human genome is also shown.
Chattopadhyay
P
P
Department of Biochemistry, All India Institute of Medical Sciences, New Delhi.
Sinha
S
S
D002412:Cations; D004247:DNA / Q000032:analysis*; D018628:Gene Dosage; D015894:Genome, Human; D006801:Humans; D008163:Luminescent Measurements; D008567:Membranes, Artificial; D009757:Nylons; D012680:Sensitivity and Specificity
D002412:Cations; D008567:Membranes, Artificial; D009757:Nylons; D004247:DNA