Palindromic sequence-targeted (PST) PCR: a rapid and efficient method for high-throughput gene characterization and genome walking

Ruslan Kalendar,Frederick L Stoddard,Alan H Schulman,Mervi M Seppänen,Alexandr V Shustov

doi:10.1038/s41598-019-54168-0

Ruslan Kalendar, Frederick L Stoddard + Show 3 more

Open Access

https://doi.org/10.1038/s41598-019-54168-0

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Abstract

Genome walking (GW) refers to the capture and sequencing of unknown regions in a long DNA molecule that are adjacent to a region with a known sequence. A novel PCR-based method, palindromic sequence-targeted PCR (PST-PCR), was developed. PST-PCR is based on a distinctive design of walking primers and special thermal cycling conditions. The walking primers (PST primers) match palindromic sequences (PST sites) that are randomly distributed in natural DNA. The PST primers have palindromic sequences at their 3′-ends. Upstream of the palindromes there is a degenerate sequence (8–12 nucleotides long); defined adapters are present at the 5′-termini. The thermal cycling profile has a linear amplification phase and an exponential amplification phase differing in annealing temperature. Changing the annealing temperature to switch the amplification phases at a defined cycle controls the balance between sensitivity and specificity. In contrast to traditional genome walking methods, PST-PCR is rapid (two to three hours to produce GW fragments) as it uses only one or two PCR rounds. Using PST-PCR, previously unknown regions (the promoter and intron 1) of the VRN1 gene of Timothy-grass (Phleum pratense L.) were captured for sequencing. In our experience, PST-PCR had higher throughput and greater convenience in comparison to other GW methods.

Highlights

Genome walking (GW) refers to a collection of methods that capture unknown genomic regions that are contiguous with and adjacent to a known DNA sequence
The cornerstone of the palindromic sequence-targeted PCR (PST-PCR) technology is a specific design of primers, the palindromic sequence-targeted (PST) primers, which are capable of annealing to uncharacterized sequences
We have described a novel PCR-based technique for capturing unknown sequences from whole-genome templates

Summary

Introduction

Genome walking (GW) refers to a collection of methods that capture unknown (unsequenced) genomic regions that are contiguous with and adjacent to a known DNA sequence. The GenomeWalker Kits are ligation-based, requiring ligation of adaptors to blunt-ended fragments of target DNA; the approach is called “suppression PCR” because the generation of side products is suppressed during PCR. The method has been designed to inhibit amplification of unwanted (Random Amplified Polymorphic DNA (RAPD) like) products that have the adaptor primer at both ends. For this purpose, it relies both on the 3′-aminated adaptor described above being present in one strand, and formation during the PCR annealing step of a “panhandle” structure that diminishes (suppresses) annealing of the adaptor primer on the other strand ( the name “suppressive PCR”)

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Conclusion