ddNTP Sequence Detection Without Polymerase-Induced False Positives
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Solution Overview
Problem
The binding of ddNTPs to double-stranded polynucleotides in the presence of a polymerase leads to false positives in sequencing and SNP detection methods, affecting melting temperature readings and causing false FRET effects.
Innovation Solution
Prevent ddNTP binding by inactivating or removing the polymerase, dephosphorylating or removing ddNTPs, outcompeting with other ddNTPs, or using polynucleotides with single-stranded regions to reduce binding, particularly through 3′ or 5′ protruding sequences or mismatches.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If ddNTPs are used in the presence of polymerase for sequencing or SNP detection, then chain termination and base identification are achieved, but false positives and false FRET effects occur due to unwanted binding to double-stranded polynucleotides
Solution Approach 1:
The patent removes the polymerase enzyme from the reaction mixture after the initial extension step, eliminating the source of unwanted ddNTP binding to double-stranded polynucleotides. This extraction of the harmful element (polymerase) prevents false positives while preserving the desired chain termination function that occurred during the extension phase.
Solution Approach 2:
The patent performs the chain extension reaction with ddNTPs and polymerase in a first step to incorporate the labeled ddNTPs at the correct positions, then removes the polymerase before the detection phase. This preliminary action ensures that the ddNTPs are properly incorporated while eliminating the risk of false binding during subsequent steps.
2Productivity
If polymerase is present during detection, then chain extension occurs, but melting temperature readings are affected and false FRET effects are generated
Solution Approach 1:
The patent extracts the polymerase from the reaction mixture before performing melting curve analysis or FRET detection. This removal eliminates the polymerase's interfering effects on Tm readings and FRET signals, ensuring that measurements reflect only the hybridization state of the polynucleotides without enzymatic interference.
3Quantity of substance
If ddNTPs bind to double-stranded polynucleotides, then signal detection is enhanced, but false positives are generated that compromise result accuracy
Solution Approach 1:
The patent removes the polymerase after the extension reaction, eliminating the catalyst that enables unwanted ddNTP binding to double-stranded polynucleotides. This ensures that any ddNTP binding observed during detection is specific to the intended target sequence rather than non-specific polymerase-mediated binding.
Solution Approach 2:
The patent completes all ddNTP incorporation in a preliminary extension step before removal of the polymerase, ensuring that specific binding events are established before the detection phase begins. This timing separation allows specific signal generation while preventing false positives during analysis.
Data Source
AI summary
The present invention relates to a method which prevents undesirable binding of ddNTPs to double stranded polynucleotides when in the presence of a polymerase. Such methods may be used to prevent the appearance of false positives in methods employing ddNTPs, e.g. in sequence detection methods. The present invention also provides a method of avoiding a false Tm reading or false FRET effects (such as false positive quenching), for example in a melting curve analysis method. In particular a method is provided in which a target nucleotide sequence in a test polynucleotide is detected using a method in which a double stranded molecule is generated which may or may not comprise two labels depending on whether the target sequence is present in which the presence of the two labels is determined, preferably by performing a melting curve analysis.