Arbitrarily Suffixed Sequence-Specific Primer PCR for Reliable Genome Walking: Taking Genome Walkings of Levilactobacillus brevis And Rice as Examples.

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: The practicability of ASP-PCR was confirmed by obtaining unknown DNAs adjacent to Oryza sativa hygromycin gene and Levilactobacillus brevis 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.