Experimental Analysis of XPCR-based protocols

TL;DR

This paper experimentally validates the XPCR protocol for gene concatenation, demonstrating its effectiveness with two genes and a three-gene permutation library, while discussing its limitations and potential applications.

Contribution

It provides a comprehensive experimental analysis of XPCR, confirming its ability to concatenate genes and exploring its limitations in gene amplification processes.

Findings

XPCR successfully concatenates two genes and a three-gene permutation library.

Overlap concatenation of multiple copies of the same gene is not feasible with XPCR.

The protocol's limits and potential are discussed based on experimental conditions.

Abstract

This paper reports some experimental results validating in a broader context a variant of PCR, called XPCR, previously introduced and tested on relatively short synthetic DNA sequences. Basic XPCR technique confirmed to work as expected, to concatenate two genes of different lengths, while a library of all permutations of three different genes (extracted from the bacterial strain Bulkolderia fungorum DBT1) has been realized in one step by multiple XPCR. Limits and potentialities of the protocols have been discussed, and tested in several experimental conditions, by aside showing that overlap concatenation of multiple copies of one only gene is not realizable by these procedures, due to strand displacement phenomena. In this case, in fact, one copy of the gene is obtained as a unique amplification product.