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Detection of Mutations in Human Cancer Using Nonisotopic RNase Cleavage Assay

  • Marianna Goldrick
  • James Prescott
Part of the Methods in Molecular Medicine book series (MIMM, volume 68)

abstract

Nonisotopic RNase cleavage assay (NIRCA) is an RNase-cleavage-based method for mutation scanning that detects mutations as double-stranded cleavage products in duplex RNA targets. A central requirement for NIRCA is the ability to produce large amounts of double-stranded target RNA by in vitro transcription of PCR products containing opposable T7 phage promoters. The target regions are amplified from genomic DNA or cDNA using forward and reverse primers with T7 promoters added to their 5′ ends. The crude polymerase chain reaction (PCR) products are then converted into double-stranded RNA by in vitro transcription with T7 polymerase, in reactions containing high concentrations of all four ribonucleotide triphosphates. Transcription typically results in a further amplification of the target region of at least 20-fold (see Fig. 1). After transcription, the reactions are heated and cooled to permit dena-turation and hybridization of the complementary strands; mutations in the target region result in base pair mismatches in the duplex RNA, from hybridization of complementary wild-type and mutant transcripts. For heterozygous samples, the endogenous wild-type allele provides the reference strand needed to create the mismatch. For homozygous samples, wild-type and experimental PCR products are mixed before transcription to create the mismatches. After hybridization, the duplex RNA targets are treated with single-strand-specific ribonucleases to cleave mismatches on both strands. The reaction products are separated by electrophoresis on native agarose gels and detected by ethidium staining. Experimental samples are scord positive for mutations if they contain smaller fragments that are not present in a no-mismatch control.
Fig. 1.

Assessing efficiency of the transcription reaction. Target regions of approx 600 bp from the human pu53 tumor suppressor gene (samples #1) and from the Factor IX clotting factor gene (samples #2) were amplified with incorporation of opposable T7 promoters as described in the text. Left-hand side of gel shows 2 μL of each PCR product from indicated samples. Lanes marked ø are no-template PCR controls. The right-hand side of the gel shows 8 μL the corresponding transcription reactions from each PCR product. The transcription reactions contained 2 μL of PCR product as template. Note the increase in ethidium-staining material seen after transcription. The ethidium-staining material trailing upward toward the wells in lanes containing transcription reactions may be owing to aberrant migration caused by the 3′ overhangs in the duplex RNA (since transcription starts at the eighteenth base and runs off after transcribing the complement of the promoter at the opposite end), or may be due to excess of transcript derived from one strand. Lane MW contains molecular size markers, Sau3 A restriction fragments of pUC 19 plasmid, with sizes in base pairs indicated.

Keywords

Polymerase Chain Reaction Product Target Region Cleavage Product Polymerase Chain Reaction Template Polymerase Chain Reaction Step 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Marianna Goldrick
    • 1
  • James Prescott
    • 2
  1. 1.Ambion RNA DiagnosticsAustin
  2. 2.UroCor, Inc.Oklahoma City

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