Screening of differentially amplified cDNA products from RNA arbitrarily primed PCR fingerprints using single strand conformation polymorphism (SSCP) gels.
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.
Mathieu-Daudé
F
F
Sidney Kimmel Cancer Center, La Jolla, CA 92037, USA.
Cheng
R
R
Welsh
J
J
McClelland
M
M
D000180:Acrylic Resins; D000818:Animals; D001483:Base Sequence; D016172:DNA Fingerprinting; D017931:DNA Primers; D018076:DNA, Complementary / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000302:isolation & purification; D005784:Gene Amplification; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D012313:RNA; D016053:RNA, Protozoan / Q000302:isolation & purification; D014346:Trypanosoma brucei brucei / Q000235:genetics*
D000180:Acrylic Resins; D017931:DNA Primers; D018076:DNA, Complementary; D016054:DNA, Protozoan; D016053:RNA, Protozoan; C016680:polyacrylamide gels; D012313:RNA