Arbitrarily Suffixed Sequence-Specific Primer PCR for Reliable Genome Walking: Taking Genome Walkings of Levilactobacillus brevis And Rice as Examples.
Year: 2024
Type: article
Abstract: Background: Genome walking has contributed to life science-related areas. Herein, we detailed a new genome walking method, nominated as arbitrarily suffixed sequence-specific primer PCR (ASP-PCR). Objectives: This study aimed to construct an efficient random PCR-based genome walking method. Materials and Methods: The key for this method is the use of a hybrid primer (HP) in primary ASP-PCR. This HP is fabricated by suffixing an arbitrary sequence to outmost sequence-specific primer. The relaxed cycle in primary ASP-PCR facilitates the partial annealing of HP to genome, creating many single-stranded DNAs. In the next stringent cycles, target single-strand is exponentially amplified, because it also has a site complementary to the sequence-specific part of HP; while nontarget cannot be further processed due to lacking such a site. Nested secondary/tertiary ASP-PCR further selectively enriches target DNA. Results: CD0817 L-glutamic acid decarboxylase gene. The results showed that each secondary or tertiary ASP-PCR exhibited 1 - 2 clear target amplicon(s) with size from 1.5 to 3.5 kb, and a weak background. Conclusions: The ASP-PCR is a promising genome walking scheme, and may have a potential use in life science-related areas.
Source: PubMed
Authors: Haixing Li, Cheng Wei, Zhenkang Pan, Xiaohua Liu
Institution: Nanchang University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: G1845091498, G6688124112
OpenAlex ID: W4409445017