• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of narLink to Publisher's site
Nucleic Acids Res. Apr 15, 1996; 24(8): 1504–1507.
PMCID: PMC145820

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.

Full Text

The Full Text of this article is available as a PDF (64K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Welsh J, Chada K, Dalal SS, Cheng R, Ralph D, McClelland M. Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 1992 Oct 11;20(19):4965–4970. [PMC free article] [PubMed]
  • Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992 Aug 14;257(5072):967–971. [PubMed]
  • Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 1990 Dec 25;18(24):7213–7218. [PMC free article] [PubMed]
  • Welsh J, Petersen C, McClelland M. Polymorphisms generated by arbitrarily primed PCR in the mouse: application to strain identification and genetic mapping. Nucleic Acids Res. 1991 Jan 25;19(2):303–306. [PMC free article] [PubMed]
  • Williams JG, Hanafey MK, Rafalski JA, Tingey SV. Genetic analysis using random amplified polymorphic DNA markers. Methods Enzymol. 1993;218:704–740. [PubMed]
  • Wong KK, McClelland M. Stress-inducible gene of Salmonella typhimurium identified by arbitrarily primed PCR of RNA. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):639–643. [PMC free article] [PubMed]
  • McClelland M, Ralph D, Cheng R, Welsh J. Interactions among regulators of RNA abundance characterized using RNA fingerprinting by arbitrarily primed PCR. Nucleic Acids Res. 1994 Oct 25;22(21):4419–4431. [PMC free article] [PubMed]
  • Ralph D, McClelland M, Welsh J. RNA fingerprinting using arbitrarily primed PCR identifies differentially regulated RNAs in mink lung (Mv1Lu) cells growth arrested by transforming growth factor beta 1. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10710–10714. [PMC free article] [PubMed]
  • Momiyama T, Afele JC, Saito T, Kayano T, Tabei Y, Takaiwa F, Takayanagi K, Nishimura S. Differential display identifies developmentally regulated genes during somatic embryogenesis in eggplant (Solanum melongena L.). Biochem Biophys Res Commun. 1995 Aug 15;213(2):376–382. [PubMed]
  • Utans U, Liang P, Wyner LR, Karnovsky MJ, Russell ME. Chronic cardiac rejection: identification of five upregulated genes in transplanted hearts by differential mRNA display. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6463–6467. [PMC free article] [PubMed]
  • Murphy NB, Pellé R. The use of arbitrary primers and the RADES method for the rapid identification of developmentally regulated genes in trypanosomes. Gene. 1994 Apr 8;141(1):53–61. [PubMed]
  • McClelland M, Mathieu-Daude F, Welsh J. RNA fingerprinting and differential display using arbitrarily primed PCR. Trends Genet. 1995 Jun;11(6):242–246. [PubMed]
  • Hayashi K. PCR-SSCP: a simple and sensitive method for detection of mutations in the genomic DNA. PCR Methods Appl. 1991 Aug;1(1):34–38. [PubMed]
  • Cully DF, Ip HS, Cross GA. Coordinate transcription of variant surface glycoprotein genes and an expression site associated gene family in Trypanosoma brucei. Cell. 1985 Aug;42(1):173–182. [PubMed]
  • Alexandre S, Guyaux M, Murphy NB, Coquelet H, Pays A, Steinert M, Pays E. Putative genes of a variant-specific antigen gene transcription unit in Trypanosoma brucei. Mol Cell Biol. 1988 Jun;8(6):2367–2378. [PMC free article] [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...