Reamplification of differential display products: more is not better.
Differential display polymerase chain reaction is technically challenging at a number of steps, including reliable reamplification of differentially expressed sequences after they are isolated from an acrylamide gel. As the only source of the sequence found to be of interest is the gel from which it was identified as being differentially expressed, failed attempts at reamplification can be particularly frustrating. In our laboratory, attempts to reamplify DNA sequences cut from a denaturing 6% acrylamide gel consistently failed when greater than 5 microliters of eluted product was used as template in a subsequent PCR reaction. If less template was used in subsequent PCR reactions, reamplification was consistently successful. This observation emphasized the importance of using limited amounts of template when reamplifying sequences that are differentially displayed on denaturing acrylamide gels
Domachowske
J B
JB
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10 Room 11N112, 9000 Rockville Pike, Bethesda, MD 20892, USA. jdomach@helix.nih.gov
Malech
H L
HL
D004247:DNA / Q000032:analysis; D020869:Gene Expression Profiling; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D004247:DNA