HBV Surface Gene Mutation Detection via Protein Size Comparison
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods fail to effectively detect C-terminal truncation mutations of the hepatitis B virus (HBV) surface gene, which are associated with hepatocellular carcinoma, as these mutations are often lost during the oncogenic process, making it difficult to identify their role in cancer initiation.
Innovation Solution
A method involving two sets of primers for PCR, followed by in vitro translation, to detect C-terminal truncation mutations of the HBV surface gene by comparing the size of the protein products with wild-type small S protein, using a FLAG tag and T7 or Sp6 promoter sequences to label and amplify the PCR products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional sequencing methods are used to detect HBV surface gene mutations, then detection accuracy is improved, but detection cost and complexity increase significantly
Solution Approach 1:
The patent creates a copy of the target HBV surface gene sequence by amplifying it through PCR using specific primers. This amplified copy contains the mutation information and can be detected through simpler means (protein expression and size comparison) rather than directly sequencing the original DNA, thus reducing complexity while maintaining detection accuracy
Solution Approach 2:
The patent replaces the mechanical/chemical process of DNA sequencing with a biological expression system. Instead of directly analyzing DNA sequences through complex sequencing machinery, the amplified DNA is transcribed and translated into proteins, and mutations are detected by comparing protein sizes through simpler biochemical methods like SDS-PAGE
2Measurement precision
If complete HBV genome sequencing is performed to identify mutations, then all mutations are detected, but time and cost consumption increase
Solution Approach 1:
The patent extracts only the specific S gene region of the HBV genome that contains the surface antigen coding sequence. By using targeted PCR primers that bind specifically to this region, the method isolates and amplifies only the relevant portion of the genome, eliminating the need to sequence the entire HBV genome while still detecting all mutations within the S gene region
Solution Approach 2:
The patent segments the HBV genome analysis into a specific focused region (the S gene). The detection process is divided into discrete steps: targeted PCR amplification of the S gene, followed by protein expression and size-based mutation detection. This segmentation allows comprehensive mutation detection within the S gene without the time burden of complete genome sequencing
3Measurement precision
If multiple PCR amplification steps are performed to amplify the target sequence, then detection sensitivity is improved, but process complexity increases
Solution Approach 1:
The patent merges the amplification and labeling functions into a single integrated PCR process. The primers used in the first PCR round not only amplify the target sequence but also incorporate the FLAG tag sequence and promoter elements. This merged approach achieves high detection sensitivity through amplification while reducing process complexity by eliminating the need for separate labeling steps
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 allows for the cost-effective detection of C-terminal truncation mutations without the need for sequencing, enabling the identification of oncogenic activity in HBV-related HCC, with the W182 mutant showing the highest incidence and tumor growth rate in xenograft studies.
Implementation Method 1
the second forward primer comprises, in the 5′→3′ direction, a T7 or an Sp6 promoter sequence
Implementation Method 2
transcribing the second PCR product into a protein
Implementation Method 3
the first forward primer comprises a nucleotide sequence for annealing to the 5′-end of the S gene and is labeled with a FLAG tag sequence at the 5′-end thereof
Data Source
AI summary
A method for in vitro detection of the presence of a C-terminal truncation mutation of a hepatitis B virus (HBV) surface (S) gene encoding a small S protein in an isolated nucleic acid sample is disclosed. An in vitro diagnostic kit for use in the aforementioned method is also disclosed.