Nucleic Acids Research, Vol 24, Issue 8 1504-1507, Copyright © 1996 by Oxford University Press
F Mathieu-Daude, R Cheng, J Welsh and M McClelland
Arbitrarily primed PCR fingerprinting of RNA and differential display
resolved on an acrylamide gel has been extensively used to detect
differentially expressed RNAs. However, after a differentially amplified
product is detected the next steps are labor-intensive: a small portion of
the fingerprinting gel that contains the differentially amplified product
is cut out, reamplified and the correct product is determined, typically by
cloning and sequencing what is often a mixture of products of similar size.
Here we use a native acrylamide gel to separate DNAs in the reamplified
mixture based on single-stranded conformation polymorphisms.
Reamplifications are performed for the region carrying the differentially
amplified product and a corresponding region from an adjacent lane where
the product is less prominent or not visible. Denaturation of the
reamplified DNA followed by side-by-side comparison on an SSCP gel allows
the classification of reamplified material into (i) those that can be
directly cloned because the differentially amplified product is relatively
pure, (ii) those that need to be reamplified from the SSCP gel before
cloning and (iii) those that are too complex for further study. This screen
should save considerable effort now wasted on directly cloning unsuitable
products from RNA fingerprinting experiments. An example is presented of
cloning a gene differentially expressed in Trypanosoma brucei life cycle.
ARTICLES
Screening of differentially amplified cDNA products from RNA arbitrarily primed PCR fingerprints using single strand conformation polymorphism (SSCP) gels
Sidney Kimmel Cancer Center, La Jolla, CA 92037, USA.
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