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LD-PCR coupled to long-read direct sequencing: an approach for mutation detection in genes with compact genomic structures

Fleming, Nick, Maynard, Julie H., Tzitzis, Loukas, Sampson, Julian Roy ORCID: and Cheadle, Jeremy Peter ORCID: 2001. LD-PCR coupled to long-read direct sequencing: an approach for mutation detection in genes with compact genomic structures. Journal of Biochemical and Biophysical Methods 47 (1-2) , pp. 131-136. 10.1016/S0165-022X(00)00159-7

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A number of techniques have been developed as primary screens to scan for DNA sequence variants, including denaturing gradient gel electrophoresis, denaturing high-performance liquid chromatography, single-strand conformation polymorphism and heteroduplex analysis. Variant alleles detected by these assays are subsequently characterised by DNA sequencing. Sequencing itself is rarely used as a primary screen because of labour intensity, cost, and, upon automation, occasional inaccuracy in identifying heterozygous sites. We have previously developed an approach based on coupling long-distance PCR (LD-PCR) to long-read direct sequencing to allow the detection of mutations in the ∼1.1 kb exon 3 of MECP2. Our use of dye-labelled primers generated high-quality bi-directional sequence runs >650 bp and allowed easy discrimination of heterozygous bases. We now describe the application of this approach to the detection of mutations in a considerably larger 6.35 kb LD-PCR fragment spanning 10 exons (exons 32–41) of the structurally complex, but genomically compact, TSC2 gene. In a blind analysis, 15/15 previously characterised mutations were successfully identified using seven overlapping bi-directional sequencing reactions. Our approach of long-read sequencing of long-distance PCR products may allow rapid sequencing of multiple exons of compact genes and may be appropriate as a highly sensitive primary screen for mutations.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Subjects: R Medicine > R Medicine (General)
Uncontrolled Keywords: direct sequencing; long-read sequencing; LD-PCR; mutation detection
Publisher: Elsevier
ISSN: 0165-022X
Last Modified: 25 Oct 2022 09:37

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