Target Gene Amplification by PCR/RT-PCR with Specialized Epitope Primers

Radioactive handling Cell-FreeTranslation with required Radiolabeled Amino Acids

Cell-Free Translation with Engineered tRNAs

No radioactivity

Low throughput applications only


High throughput ELISA-based PTT screening (>1000 samples)

8-48 hrs for detection

Subjective Results

Not amenable to automation

Autoradiography t

Manual Readout

Chemiluminescence Detection 5 min for detection

Total Assay Time: 10-50 hrs

Automatic Readout t

Follow-up Analysis t

PAGE-based PTT t

Fluorescence Detection

Objective Results

Positive samples can be validated and truncation location identified

Amenable to automation

Total Assay Time: 1-2 hrs

Fig. 1 Various steps involved in typical protein truncation test. (View this art in color at www.dekker.com.)

In addition, several attempts have been made to scan entire genes for truncating mutations using a combination of genomic DNA and mRNA (Table 1). In the case of RNA-based PTT, mRNA is first reverse transcribed to cDNA, which then functions as a template for amplification. The best source of RNA is cells in which the target gene is abundantly expressed. However, for practical reasons RNA is generally isolated from freshly drawn peripheral blood lymphocytes.

Was this article helpful?

0 0
Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook

Post a comment