Objective: To develop a rapid, robust and more reliable method for an expeditious genetic investigation of patients with TRDs.to develop a rapid, robust and more reliable method for an expeditious genetic investigation of patients with TRDs.

Background: DNA extraction prior to PCR amplification in genetic diagnoses of triplet repeat disorders (TRDs) is tedious and labor intensive and has the limitations of sample contamination with foreign DNA and including from preceding samples. Therefore, we aimed to develop a rapid, robust and more reliable method for an expeditious genetic investigation of patients with TRDs.

Method: Peripheral blood samples of suspected patients of Spinocerebellar ataxia types 1, 2, 3, 12 and FRDA were collected in ACD buffer vials and tested for repeat expansion using conventional method. Subsequently, a single step Blood-Direct PCR (using 1.5µl whole blood of positive patients) technique was used to amplify target genetic loci followed by fragment analysis for the identification of triplet repeat expansion.

Results: Successful amplification of target regions was achieved by BD-PCR in all tested samples. The repeat sizes determined in each fragment of DNA were in concordance with the conventional method.

Conclusion: The accurate sizing of the normal and expanded allele in shorter time (4-5 hours) without DNA extraction and cross contamination suggests the BD-PCR method to be a reliable, inexpensive and rapid method. This technique can be introduced in routine diagnostic procedures of other tandem repeat disorders.

References: Singh I, Swarup V, Shakya S, Goyal V, Faruq M, Srivastava AK. Single-step blood direct PCR: A robust and rapid method to diagnose triplet repeat disorders. J Neurol Sci. 2017 Aug 15; 379:49-54.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/a-robust-and-rapid-method-of-pcr-amplification-to-confirm-triplet-repeat-disorders-single-step-blood-direct-pcr/