Generation of a 1.5‐kb cDNA fragment of the hepatitis C virus genome by overlap extension

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Abstract
Hepatitis C virus (HCV) is a recently identified RNA virus responsible for most of non‐A, non‐B hepatitis. Genetic analysis of HCV sequences and their encoded proteins has been hampered by the difficulty in obtaining cDNA fragments of sufficient lengths: construction of cDNA library requires technical expertise while amplification by the polymerase chain reaction (PCR) usually yields fragments of less than 400 base pairs. In this report we have generated a 1.5‐kb HCV cDNA fragment by overlap extension of smaller PCR fragments and by ligation through restriction sites. Sequencing of the cloned fragment confirmed the absence of significant sequence alteration produced by this procedure. Overlap extension may represent an easy method for generating relatively large HCV cDNA clones for the expression of HCV‐encoded proteins.