19 cDNA Library Screening
291
3. Phage particles are disrupted or bacteria are lysed in situ on the filters.
4. DNA is bound to the fllter while RNA is hydrolyzed.
5. Filtersare washed and non-specific binding sites are blocked during a
prehybridization step. The bound partiallydenatured DNA is hybridized
to a labeled complementary nucleic acid sequence probethat is capable
of binding to the immobilized target sequences at the experimentally
chosen hybridization stringency.
6. The pattern of the DNA on the filter replicas is aligned with the plated
recombinants on the masters. The image of positive signals obtained,
therefore, serves as a map for picking positive recombinants from the
master.
7. Positive colonies or plaques are purified and reamplified for further analysis.
To avoid false positive clones, which are quite common using this method, it
is highly recommended that duplicate filters of each master plate are
screened in addition to negative and positive controls. Hybridization
screening requires information about the sequence being sought. Often
part of the sequence to be isolated is already known and the procedure
is used to search for additional flanking sequence regions. In other cases,
closely related sequences may cross-hybridize to the desired cDNA and can
be used as probes. For optimal screening results, careful consideration of
hybridization parameters is required. For DNA-DNA interactions these are
related as follows (Wahl et al. 1987): Tm= 81.5° + 16.6logM + 41 (o/o[G+C]500/L-0.62 (o/oformamide) where T rn is the melting temperature, defined as
the temperature at which half the duplex molecules have dissociated into
their constituent single strands; M is the monovalent cation concentration
in moles per liter; ( G+C) is the base composition expressedas mole fraction
of G and C residues; L is the length of the shortest chain in the duplex and
%formamide is the relative concentration of the helix-destabilizing agent.
The maximum rate of hybridization occurs at 25° below Tm with a broad
range of nearly ideal conditions between Tm -32° and Tm -18°. Standard
conditions used are Tm -25°, 0.18M cation concentration, and a single
stranded fragment length of 500 nucleotides. Formostexperiments this results in optimal hybridization temperatures between 65°C to 68°C, or approx. 42°C in the presence of 50% formamide.
Base mismatches decrease the stability of hybrid duplexes according to
mismatch number and location. For hybrids Ionger than 150 bp, the Tm of a
DNA duplex decreases by approx. 1 o for every 1% ofbase pair mismatch. In
291
3. Phage particles are disrupted or bacteria are lysed in situ on the filters.
4. DNA is bound to the fllter while RNA is hydrolyzed.
5. Filtersare washed and non-specific binding sites are blocked during a
prehybridization step. The bound partiallydenatured DNA is hybridized
to a labeled complementary nucleic acid sequence probethat is capable
of binding to the immobilized target sequences at the experimentally
chosen hybridization stringency.
6. The pattern of the DNA on the filter replicas is aligned with the plated
recombinants on the masters. The image of positive signals obtained,
therefore, serves as a map for picking positive recombinants from the
master.
7. Positive colonies or plaques are purified and reamplified for further analysis.
To avoid false positive clones, which are quite common using this method, it
is highly recommended that duplicate filters of each master plate are
screened in addition to negative and positive controls. Hybridization
screening requires information about the sequence being sought. Often
part of the sequence to be isolated is already known and the procedure
is used to search for additional flanking sequence regions. In other cases,
closely related sequences may cross-hybridize to the desired cDNA and can
be used as probes. For optimal screening results, careful consideration of
hybridization parameters is required. For DNA-DNA interactions these are
related as follows (Wahl et al. 1987): Tm= 81.5° + 16.6logM + 41 (o/o[G+C]500/L-0.62 (o/oformamide) where T rn is the melting temperature, defined as
the temperature at which half the duplex molecules have dissociated into
their constituent single strands; M is the monovalent cation concentration
in moles per liter; ( G+C) is the base composition expressedas mole fraction
of G and C residues; L is the length of the shortest chain in the duplex and
%formamide is the relative concentration of the helix-destabilizing agent.
The maximum rate of hybridization occurs at 25° below Tm with a broad
range of nearly ideal conditions between Tm -32° and Tm -18°. Standard
conditions used are Tm -25°, 0.18M cation concentration, and a single
stranded fragment length of 500 nucleotides. Formostexperiments this results in optimal hybridization temperatures between 65°C to 68°C, or approx. 42°C in the presence of 50% formamide.
Base mismatches decrease the stability of hybrid duplexes according to
mismatch number and location. For hybrids Ionger than 150 bp, the Tm of a
DNA duplex decreases by approx. 1 o for every 1% ofbase pair mismatch. In