Direct cDNA Sequencing via dUTP Cleavage and Circularization
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Solution Overview
Problem
Existing gene expression analysis methods face challenges in accurately quantifying rare DNA and mRNA/cDNA molecules due to uneven PCR amplification and inefficient removal of unincorporated primers, leading to suboptimal sequencing coverage.
Innovation Solution
A method involving the incorporation of dUTP into DNA, followed by cleavage at U sites, cyclization of DNA fragments, and subsequent sequencing, which allows for direct sequencing without prior amplification and provides unbiased quantification of nucleic acid molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If PCR amplification is used to generate DNA libraries for sequencing, then sufficient DNA quantity is obtained for sequencing, but uneven coverage and unreliable quantification of rare molecules occur
Solution Approach 1:
The invention extracts and removes the harmful PCR amplification step from the library preparation process. By using direct reverse transcription followed by dUTP incorporation and enzymatic cleavage, the method obtains sufficient DNA quantity for sequencing without the amplification bias that compromises quantification accuracy of rare molecules
Solution Approach 2:
The invention changes the chemical parameter of nucleotide incorporation by using dUTP instead of dTTP during reverse transcription. This parameter change enables subsequent enzymatic cleavage at dU sites, allowing direct sequencing library generation without PCR amplification, thereby maintaining both sufficient DNA quantity and accurate quantification
2Quantity of substance
If PCR amplification is used to generate DNA libraries, then sufficient DNA quantity is obtained, but the sequencing coverage becomes uneven
Solution Approach 1:
The invention removes the PCR amplification step that causes uneven sequencing coverage. By using direct reverse transcription with dUTP incorporation followed by enzymatic cleavage and circularization, sufficient DNA quantity is obtained while maintaining uniform sequencing coverage across all molecules
Solution Approach 2:
The invention uses reverse transcription to create cDNA copies of RNA molecules directly, without subsequent PCR amplification. This single-step copying process with dUTP incorporation ensures each original RNA molecule is represented by one cDNA copy, resulting in uniform sequencing coverage
3Object-generated harmful factors
If size-dependent separation methods like PAGE are used to remove unincorporated primers, then primer removal is achieved, but quantitative yield and discrimination between similar-sized molecules deteriorate
Solution Approach 1:
The invention introduces a local chemical difference by incorporating dUTP at specific positions within the cDNA molecules. This creates unique dU sites that can be selectively recognized and cleaved by enzymes, enabling specific processing of cDNA molecules without affecting their overall size or requiring size-dependent separation methods
Solution Approach 2:
The invention uses dU residues as intermediary markers within the cDNA molecules. These markers serve as recognition sites for enzymatic cleavage, allowing specific processing and removal of unincorporated primers without relying on size-dependent separation that compromises molecule discrimination accuracy
4Measurement precision
If molecular random identifiers are added to eliminate uneven coverage, then quantification accuracy improves, but high sequencing coverage is required which increases cost and time
Solution Approach 1:
The invention removes the need for molecular random identifiers and high sequencing coverage requirements by eliminating PCR amplification entirely. Direct reverse transcription with dUTP incorporation followed by enzymatic cleavage provides accurate quantification of rare molecules without requiring expensive and time-consuming deep sequencing
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 enables accurate determination of nucleic acid molecules at low sequencing density, improving gene expression analysis by reducing amplification bias and enhancing sequencing efficiency.
Implementation Method 1
transcribing the nucleic acid to form single-stranded DNA by contacting the nucleic acid with a DNA polymerase, a primer and a mixture of dNTPs under conditions that allow the generation of the DNA, wherein the dNTP mixture comprises dATP, dGTP, dCTP, dTTP and dUTP
Implementation Method 2
cleaving the DNA 5′ to dU sites by (i) contacting the DNA with an Uracil Deglycosylase to generate abasic sites at positions of dUTP incorporation in the DNA
Implementation Method 3
contacting the DNA with an apurinic/apyrimidinic (AP) endonuclease
Implementation Method 4
contacting the DNA comprising at its 5′-end the nucleotide sequence of the primer with a ssDNA ligase to circularize the DNA comprising at its 5′-end the nucleotide sequence of the primer
Data Source
AI summary
The present invention is directed to methods and kits for gene analysis. The methods of the invention comprise the steps of providing nucleic acid; synthesis of a single-stranded DNA that is complementary to said nucleic acid molecule by contacting the nucleic acid with a DNA polymerase, a primer and a mixture of dNTPs under conditions that allow the generation of the DNA, wherein the primer comprises a target-complementary region and wherein the dNTP mixture comprises dATP, dGTP, dCTP, dTTP and dUTP; cleaving the DNA 5′ to dU sites by (i) contacting the DNA with an uracil deglycosylase to generate a basic sites at positions of dUTP incorporation in the DNA; and (ii) contacting the DNA with an apurinic/apyrimidinic (AP) endonuclease; contacting the DNA comprising at its 5′-end the nucleotide sequence of the primer with a ssDNA ligase to circularize the DNA; and sequencing the circularized cDNA. The kits comprise the components necessary to perform the methods of the invention.