A simple, effective method for the construction of subtracted cDNA libraries.
A simple method is described for the construction of subtracted cDNA libraries. The technique was used to create a human pancreatic tumor cDNA library that was screened using either hybridization with cDNA probes or antibodies. cDNA from a well-differentiated tumor cell line (CD-11) was subtracted against RNA from an undifferentiated tumor cell line (Panc-1). The subtracted cDNA was purified from RNA-cDNA hybrids by oligo-dA cellulose affinity chromatography. Single-stranded subtracted cDNA was used as a template for random primed second-strand synthesis using the Klenow's fragment of DNA polymerase. After ligation with Eco R1 adapters, cDNA was inserted into lambda gt11. A library of 140,000 primary pfu was obtained that contained 92% recombinants. A small portion of this library (40,000 pfu) was subjected to probe screening with a mucin cDNA probe known to be differentially expressed by CD-11 cells. The ratio of mucin cDNA clones to actin cDNA clones was increased by greater than 300-fold in the subtracted cDNA library compared to a standard cDNA library from the same cell line. The absolute number of mucin cDNA clones per 40,000 pfu was also increased 32-fold in the subtracted library. Pancreatic tumor mucin cDNAs were also identified in the subtracted library by antibody screening. The subtraction procedure yielded a 50-fold enrichment in differentially expressed cDNA detected by antibodies, compared to a nonsubtracted library from the same cell line.
Batra
S K
SK
Duke University Medical Center, Durham, North Carolina.
Metzgar
R S
RS
Hollingsworth
M A
MA
D000906:Antibodies; D002460:Cell Line; D002999:Clone Cells; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded / Q000302:isolation & purification*; D015723:Gene Library; D005821:Genetic Techniques; D006801:Humans; D009077:Mucins / Q000235:genetics*; D009693:Nucleic Acid Hybridization
D000906:Antibodies; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D009077:Mucins