Human cDNA clones represent recombinant DNA molecules containing complementary DNA (cDNA) sequences synthesized from human messenger RNA (mRNA), capturing protein-coding genes without introns for functional studies and expression.
Definition and Synthesis
cDNA clones derive from reverse transcription of poly(A)+ mRNA using reverse transcriptase, yielding first-strand cDNA extended by DNA polymerase for double-stranded molecules, then ligated into vectors such as plasmids or lambda phage. Human libraries from tissues (brain, liver) or cell lines provide full-length or partial clones, often with 5'/3' UTRs. Collections like hORFeome or CCSB-Broad encompass more than 30,000 verified human ORFs in Gateway-compatible entry vectors.
Characteristics
Clones feature correct reading frames (70% in high-quality libraries), His-tags for purification, sizes ranging from 0.5-10 kb matching predicted proteins (15-200 kDa), and verified inserts via Sanger or next-generation sequencing. Full-length clones prioritize complete ORFs, splicing variants capture isoforms, and solubility screening identifies E. coli-expressible proteins (~25% success rate).
Construction and Screening
Libraries array 105-106 clones on membranes for immunological detection of His-fusions, followed by small-scale expression and purification. For example, 2,746 out of 10,825 clones from the hEx1 library expressed soluble proteins ≥15 kDa. Transfer to lentiviral, baculoviral, or mammalian vectors (e.g., pcDNA) enables overexpression. Structural genomics selects clones for crystallization (e.g., 17 out of 163 yielded 3 structures).
Applications
Human cDNA clones facilitate protein production for X-ray crystallography and NMR, functional assays, high-throughput arrays, and gene therapy vectors. They define isoforms (e.g., N-CAM 120/140/180 kDa via splicing) and support transcriptomics studies.
