Adjacent Sequence PCR for Sensitive DNA Insertion Site Mapping
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Solution Overview
Problem
Current methods for identifying the insertion site of foreign DNA on the host genome are inefficient, costly, and lack sensitivity, particularly in gene therapy and genome editing, posing risks due to unknown integration sites.
Innovation Solution
A method involving steps 1 to 6 for amplifying a sequence adjacent to a specific sequence, utilizing primers and polymerase chain reactions to achieve high sensitivity, efficiency, and cost-effectiveness, applicable to DNA and RNA strands.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods (mate pair sequences, inverse PCR, Tail-PCR, LAM-PCR, nrLAM-PCR) are used to identify insertion sites of foreign DNA, then the identification can be performed, but the methods are insensitive, costly, and require excessive time
Solution Approach 1:
The invention divides the identification process into two distinct amplification stages: first amplifying the junction region between foreign DNA and host genome using foreign DNA-specific primers, then amplifying the adjacent host genome sequence using nested primers. This segmented approach increases sensitivity by focusing on specific regions rather than attempting to amplify the entire genome in one step.
Solution Approach 2:
The method performs preliminary amplification of the junction region before amplifying the adjacent sequence. By first enriching the junction region containing the insertion site information, the subsequent amplification of adjacent sequences becomes more efficient and sensitive, avoiding the need to process entire genomic DNA.
2Measurement precision
If conventional methods are used, then insertion site identification is possible, but the cost is high and operability is poor
Solution Approach 1:
The invention extracts only the necessary information by using foreign DNA-specific primers to amplify only the junction region containing insertion site data, rather than sequencing or analyzing entire genomic DNA. This extraction approach reduces cost while maintaining identification accuracy.
Solution Approach 2:
The method creates multiple copies of the target junction region and adjacent sequences through PCR amplification, generating sufficient material for analysis without requiring expensive whole-genome sequencing. The nested PCR approach produces enriched copies of specific regions of interest.
3Area of stationary object
If genome-wide methods are used, then comprehensive coverage is achieved, but selectivity is lacking and sensitivity decreases
Solution Approach 1:
The invention applies local quality by designing primers with specific properties for different regions: foreign DNA-specific primers target the junction region with high specificity, while nested primers target adjacent host genome sequences. This localized optimization of primer specificity enhances sensitivity for insertion site detection without requiring comprehensive genomic coverage.
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
Enables rapid amplification and sequencing of adjacent sequences with high sensitivity, reproducibility, and reduced costs, suitable for various genomic DNAs and RNA strands, including HTLV-1, HIV-1, SIV, HBV, and adenovirus insertion sites.
Implementation Method 1
a first forward primer is annealed to a specific sequence
Implementation Method 2
performing an extension reaction with the primer as a starting point, and synthesizing a complementary strand
Implementation Method 3
polymerically adding a deoxynucleotide to a 3'-end of the complementary strand obtained in step (1)
Implementation Method 4
a first reverse primer is annealed to a binding site between the 3'-end of the complementary strand and the polydeoxynucleotide strand
Implementation Method 5
performing an extension reaction with the primer as a starting point, and synthesizing a double-stranded DNA
Implementation Method 6
performing a polymerase chain reaction with the double-stranded DNA synthesized in step (4) as a template
Data Source
AI summary
The present invention provides a method for amplifying a sequence adjacent to a specific sequence, comprising the steps of: annealing a first forward primer to the specific sequence to synthesize a complementary strand; sequentially polymerically adding a first deoxynucleotide and a second deoxynucleotide to a 3′-end of the complementary strand; annealing a first reverse primer to a binding site between the 3′-end of the complementary strand and a polydeoxynucleotide strand composed of the first deoxynucleotide to synthesize a double-stranded DNA; performing a PCR with the double-stranded DNA as a template by using a second forward primer complementary to the specific sequence and a first reverse primer; and further performing a PCR by using a third forward primer complementary to the specific sequence and a second reverse primer.