Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 65)

Fig. 6.6. Pouring a SSCP gel.
4. Pour or pipette the gel solution into the sandwich until it covers the wells of the comb.
Straighten the comb to the desired well depth. Add more solution if needed.
5. Allow the gel to polymerize for about 60 minutes. After polymerization, remove the comb
by pulling it straight up slowly and gently.
6. Continue with Section 8 for electrophoresis.
Section 7
Protein Truncation Test
7.1 Introduction to PTT
Increasing numbers of genes with translation terminating mutations are being identified.
The Protein Truncation Test (PTT) is a mutation screening method that detects truncated
proteins after translation of the coding sequence.
7, 30 There are six steps associated with the
PTT assay. The first step is to amplify by PCR a template RNA sample. The second step
requires a reverse transcriptase reaction of the starting mRNA to make cDNA. The third
step amplifies the sequence of interest and incorporates a tailed primer sequence. This
tailed primer contains a T7 promoter and eukaryotic translation initiation sequence. These
sequences are needed for in vitro transcription and translation. The fourth step checks the
PCR product on an agarose gel for quality, size, and approximate quantity. In the fifth step,
the PCR products are transcribed with RNA polymerase and translated into peptides. There
are commercial kits that couple the transcription and translation reaction in one tube using
rabbit reticulocyte lysate (Promega). Detection of the translation products is done by adding
radiolabeled amino acids, typically
3 H-Leucine or 35 S-methionine, to the translation reaction.
The final step involves analyzing the translation products on an SDS-PAGE gel to determine
their length. The gel is normally treated with a fluorographic-enhancing reagent to reduce
the exposure time to X-ray film. Truncated proteins are identified by size differences when
compared to full-length control proteins.
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