18 cDNA Libraries
275
the the oligo(dT) as the solution slowly drips through the column under
gravity. Non-poly(A)+ RNA does not bind and is easily washed off the
column. The bound mRNA is rapidly eluted by washing the column with
a low-salt buffer. In a modification of this technique [ oligo( dT) spin columns], centrifugation replaces gravity as the force separating the components. Consequently, the time allowed for annealing and washing is
shortened. The total time required to isolate mRNA is reduced from several hours to less than 1 h, but yield is also reduced.
• Solution hybridization: This protocol also uses oligo(dT)-cellulose but
the matrix is added directly to the total RNA solutionrather than a column. The annealing and washing steps are performed in solution with
the matrix-bound RNA pelleted by centrifugation between steps. This
method benefits from the rapidity and efficiency of solution kinetics.
• Biotinylated oligo(dT) and streptavidin-coated magnetic beads: In this
protocol, a biotinylated oligo(dT) primer is annealed in solution to total
RNA. Magnetic beads which have been coated with streptavidin are then
added, and the bead-mRNA complex is separated from the bulk solution
using a magnet. The magnet is removed, wash solutions are added, and
the magnetic isolation is repeated. The oligo( dT) primer dissociates from
mRNA in the absence of salt. This is an extremely fast, inexpensive and
efficient method for the isolation of mRNA. In an alternative method, the
magnetic beads are directly bound to oligo(dT).
cDNA synthesis and cloning
Since mRNAs can not be propagatedas RNA molecules, they are copied into
single-stranded cDNAs by an enzyme called reverse transcriptase prior to
cloning (see Procedure). Single-stranded DNAs, however, arealso unsuitable for cloning since most ligases will only join tagether double-stranded
molecules. Thus, complementary strands are synthesized by a DNA polymerase, and the double-stranded products are cloned in cDNA libraries
(Figure 1). The efficiency in generating complete, double-stranded molecules is variable, and very short fragments often contaminate the mixture.
Moreover, shorter molecules are preferentially cloned and, if not removed,
will result in a library that is poorly representative of its source mRN A. This
problern can be avoided by size-fractionation of the cDNA prior to cloning.
This is accomplished by either agarase gel electrophoresis or centrifugation
through sizing columns that retain small molecules.
275
the the oligo(dT) as the solution slowly drips through the column under
gravity. Non-poly(A)+ RNA does not bind and is easily washed off the
column. The bound mRNA is rapidly eluted by washing the column with
a low-salt buffer. In a modification of this technique [ oligo( dT) spin columns], centrifugation replaces gravity as the force separating the components. Consequently, the time allowed for annealing and washing is
shortened. The total time required to isolate mRNA is reduced from several hours to less than 1 h, but yield is also reduced.
• Solution hybridization: This protocol also uses oligo(dT)-cellulose but
the matrix is added directly to the total RNA solutionrather than a column. The annealing and washing steps are performed in solution with
the matrix-bound RNA pelleted by centrifugation between steps. This
method benefits from the rapidity and efficiency of solution kinetics.
• Biotinylated oligo(dT) and streptavidin-coated magnetic beads: In this
protocol, a biotinylated oligo(dT) primer is annealed in solution to total
RNA. Magnetic beads which have been coated with streptavidin are then
added, and the bead-mRNA complex is separated from the bulk solution
using a magnet. The magnet is removed, wash solutions are added, and
the magnetic isolation is repeated. The oligo( dT) primer dissociates from
mRNA in the absence of salt. This is an extremely fast, inexpensive and
efficient method for the isolation of mRNA. In an alternative method, the
magnetic beads are directly bound to oligo(dT).
cDNA synthesis and cloning
Since mRNAs can not be propagatedas RNA molecules, they are copied into
single-stranded cDNAs by an enzyme called reverse transcriptase prior to
cloning (see Procedure). Single-stranded DNAs, however, arealso unsuitable for cloning since most ligases will only join tagether double-stranded
molecules. Thus, complementary strands are synthesized by a DNA polymerase, and the double-stranded products are cloned in cDNA libraries
(Figure 1). The efficiency in generating complete, double-stranded molecules is variable, and very short fragments often contaminate the mixture.
Moreover, shorter molecules are preferentially cloned and, if not removed,
will result in a library that is poorly representative of its source mRN A. This
problern can be avoided by size-fractionation of the cDNA prior to cloning.
This is accomplished by either agarase gel electrophoresis or centrifugation
through sizing columns that retain small molecules.