Subtractive hybridization system using single-stranded phagemids with directional inserts.
We describe a subtractive hybridization protocol which is designed to permit subtractions between cDNA libraries. The method uses single-stranded phagemids with directional inserts as both the driver and the target. We modified the M13 phagemid vector pBluescript for the directional cDNA cloning and subtractive hybridization. Two simplified methods for efficient construction of directional cDNA libraries are also described. Using a model system, we found that one round of subtractive hybridization results in a 5,000-fold specific subtraction of abundant molecules. We used two methods to quantify the efficiency and verify the specificity of the subtraction. In order to obtain these subtraction efficiencies, it was necessary to develop a method to purify the single-stranded DNA to homogeneity. The single-stranded purification involved using potassium iodide (KI) density centrifugation, restriction endonuclease digestion and phenol extraction in the presence of magnesium. We describe the several advantages of using directional inserts for the subtraction procedure.
Rubenstein
J L
JL
Department of Psychiatry and Behavioral Sciences, Stanford Medical School, CA.
Brice
A E
AE
Ciaranello
R D
RD
Denney
D
D
Porteus
M H
MH
Usdin
T B
TB
D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular / Q000379:methods; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D004279:DNA, Viral; D004587:Electrophoresis, Agar Gel; D015723:Gene Library; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010636:Phenols; D010957:Plasmids; D011193:Potassium Iodide; D014169:Transformation, Bacterial
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D010636:Phenols; D011193:Potassium Iodide; D004262:DNA Restriction Enzymes