Multiple specific/nonspecific primers for PCR of a complex gene pool

Abstract

Disclosed are compositions and methods for single-step PCR of a sample containing a complex target gene pool that can simultaneously amplify a wide variety of variant target gene sequences common to the sample while maintaining the original ratios of gene variants. The compositions and methods described herein utilize (1) a gene-specific primer pool that contains multiple variants that occur in asample containing a complex mixture of target sequences that are both required for amplification of variants in the mixture which may introduce amplification bias, with (2) a non-specific PCR primer that is designed to target multiple gene-specific primer variants and eliminate amplification bias.

Description

technical field [0001] Disclosed herein are compositions and methods useful for performing single-step polymerase chain reaction (PCR) of complex target gene repertoires, such as microbiome samples, using specific and non-specific primers to amplify target sequences while maintaining genetic variants and reduce or eliminate primer concentration-dependent PCR amplification bias (amplification bias). Background technique [0002] Studying the microbiome involves categorizing the types of microbes present and how many of each type there are. Targeted sequencing of 16S, 18S, 23S, internal transcribed spacer (ITS), or other combinations of conserved and variable genetic regions can be used to identify microorganisms in complex mixtures. Most targeted regions contain conserved sequences present in many domains of life that can be targeted by PCR primers that flank less conserved variable body provided a positive identification. PCR assays have the advantage that the amount of s...

AI Technical Summary

Problems solved by technology

As a result of primer depletion during PCR, the original population ratio of the target gene (representing the organism) will be distorted, artificially inflating the expressed low abundance and / or suppressing the high abundance of the organism in the microbial mixture, which will Significantly complicates interpretation of microbiome data

[0003] Missing microbial variants can be addressed by including PCR primer variants, but this introduces a new problem of primer concentration-dependent PCR amplification bias

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