✓Isolate and analyze PCR products by gel electrophoresis and BLAST sequencing
Protocol
We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1–2 copies) integration using inverse nested PCR.
Difficulty
advanced
Total time
~3–4 days (infection setup ~24 hrs, DNA extraction ~2 hrs, inverse PCR and product analysis ~2 days)
Model organism
Hepatocyte culture (in vitro HBV infection system)
Biosafety
BSL-2
Steps
1
Infect hepatocytes and extract total DNA
Culture hepatocyte cells and infect with Hepatitis B virus. After infection, extract total genomic DNA from infected cells using standard lysis and purification protocols.
▶ 00:52
2
Invert extracted DNA for PCR amplification
Digest extracted DNA with restriction enzymes and ligate the inverted DNA fragments to circularize viral sequences, enabling inverse PCR primer design and amplification across integration junctions.
▶ 02:42
3
Perform nested PCR on inverted DNA
Conduct two rounds of nested PCR using inverse primers on the inverted DNA template to amplify low-copy integrated HBV sequences with high specificity and sensitivity.
▶ 05:55
4
Isolate PCR product and purify from gel
Run PCR products on agarose gel, excise bands of expected size, and extract DNA from gel slices using standard purification methods.
▶ 08:30
5
Analyze sequences by electrophoresis and BLAST
Perform agarose gel electrophoresis to visualize purified products and sequence PCR amplicons by Sanger sequencing, then validate integrated HBV DNA identity using BLAST homology search.
▶ 09:37
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