PCR Primers With Cleavable Bases for Accurate Sequencing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current PCR methods, especially multiplex PCR, face challenges in downstream analysis due to inaccurate primer removal, leading to false-positive and false-negative variant calls and low base alignment quality scores near the ends of reads, caused by incomplete digestion of primer bases.
Innovation Solution
Designing primers with cleavable bases, such as uracil, at least 4 nucleotides away from the 3′ terminus to minimize primer digestion errors, ensuring accurate alignments and preventing misinterpretations in variant calls by maintaining a minimum distance between the cleavable base and the primer end.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If cleavable bases are placed close to the 3′ terminus of primers to minimize sequencing capacity waste, then primer removal efficiency is improved, but downstream analysis accuracy deteriorates due to incomplete digestion and false-positive/negative variant calls
Solution Approach 1:
The patent applies preliminary action by placing cleavable bases (uracil) at a specific distance (at least 4 nucleotides) from the 3′ terminus of primers before PCR amplification. This pre-positioning ensures that when UNG enzyme is added after amplification, it can efficiently digest the primers from the cleavable base location without leaving incomplete digestion products that would interfere with downstream variant calling accuracy.
2Reliability
If cleavable bases are placed at a minimum distance of 4 nucleotides from the 3′ terminus to improve downstream analysis accuracy, then variant call reliability is improved, but sequencing capacity waste increases due to retained primer bases
Solution Approach 1:
The patent applies parameter changes by optimizing the distance parameter between the cleavable base and the 3′ terminus of the primer. By setting this distance to at least 4 nucleotides, the patent finds the optimal balance that allows sufficient UNG enzyme access for complete primer digestion (improving reliability) while minimizing the number of readable bases retained in the final sequence (reducing sequencing capacity waste).
3Productivity
If traditional primer designs are used with cleavable bases near the 3′ end, then primer digestion is more efficient, but base alignment quality scores deteriorate near the ends of reads
Solution Approach 1:
By pre-positioning the cleavable base at least 4 nucleotides from the 3′ terminus, the patent ensures that UNG enzyme has adequate space to initiate and complete primer digestion before reaching the read region. This preliminary positioning prevents the enzyme from creating partial digestion products that would align poorly to the reference genome, thereby maintaining high base alignment quality scores at the ends of reads.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enhances the accuracy of downstream sequence data analysis by reducing false positives and negatives, improving base alignment quality and ensuring reliable variant detection.
Implementation Method 1
The PCR reaction proceeds as usual, incorporating Adenine (A) bases into the PCR product at positions complementary to the U bases in the primer. After the PCR step, the double stranded DNA product is treated with uracil-N-glycosylase (UNG, also known as uracil-DNA glycosylase (UDG)), which causes degradation of the primer strand until the furthest 3′ U base
Data Source
AI summary
The present disclosure provides methods, compositions, and kits for performing PCR (including multiplex PCR). The methods, compositions and kits provided herein use one or more primer pairs that contain one or more cleavable bases located at a minimal distance away from the 3′ termini of the primers, and increase the accuracy of downstream analysis of sequence data.