290
JOHANN PETER HOSSLE
Homologaus nucleic acid probes
quence during screening, it will be necessary to screen a certain number of
individual recombinants. As a rule of thumb, the number of individual recombinants in the library should exceed 5 times the number expected, given
the relative abundance ofthe desired mRNA in the original RNA population
(Kimmel and Berger 1987). lt is preferable to use unamplified libraries, because the relative frequency of recombinant clones contained in the library
may be distorted after amplification due to uneven growth rates of the individual recombinants.
Cloning of cDNA into most vectors is performed by insertion into the ßgalactosidase (lacZ) gene that is present in the vector molecule. Inserted
DNA interrupts the coding sequence in lacZ, thereby causing the phenotype
of the vector to change from blue (parental) to colorless (recombinant) in a
simple blue plaque test. In this fashion, the number of recombinants present
in the library can be determined easily. Average size A.-libraries are usually
in the range of 10 6 -10 8 plaque-forming units with around 60o/o recombinants.
Number of recombinants needed to be screened
The number of unique recombinants (N) that must be screened to ensure
isolation of the desired sequence can be calculated as: N = :~g :=~j where Pis
the probability of isolating the desired sequence ( usually set at 99o/o ); and n
is the fractional proportion of the total mRNA population represented by
the desired single mRNA species (Jendrisak et al. 1987). The abundance of
the mRNA encoding the sequence of interest needs tobe estimated by, e.g.,
quantitative RT-PCR. The numbers ofrecombinants that are required can
be us high as 10 6 for rare mRNA species.
Screening based on sequence homology
The most direct method for screening a cDNA library is to use nucleic acid
probes for hybridization. This involves the following steps (Wahl and Berger 1987):
1. Aliquots of the library tobe screened (phage or plasmid transfected bacteria) are grown on master plates (or filters) from which replica filters are
made.
2. The masters are preserved while the replica filters are processed.
JOHANN PETER HOSSLE
Homologaus nucleic acid probes
quence during screening, it will be necessary to screen a certain number of
individual recombinants. As a rule of thumb, the number of individual recombinants in the library should exceed 5 times the number expected, given
the relative abundance ofthe desired mRNA in the original RNA population
(Kimmel and Berger 1987). lt is preferable to use unamplified libraries, because the relative frequency of recombinant clones contained in the library
may be distorted after amplification due to uneven growth rates of the individual recombinants.
Cloning of cDNA into most vectors is performed by insertion into the ßgalactosidase (lacZ) gene that is present in the vector molecule. Inserted
DNA interrupts the coding sequence in lacZ, thereby causing the phenotype
of the vector to change from blue (parental) to colorless (recombinant) in a
simple blue plaque test. In this fashion, the number of recombinants present
in the library can be determined easily. Average size A.-libraries are usually
in the range of 10 6 -10 8 plaque-forming units with around 60o/o recombinants.
Number of recombinants needed to be screened
The number of unique recombinants (N) that must be screened to ensure
isolation of the desired sequence can be calculated as: N = :~g :=~j where Pis
the probability of isolating the desired sequence ( usually set at 99o/o ); and n
is the fractional proportion of the total mRNA population represented by
the desired single mRNA species (Jendrisak et al. 1987). The abundance of
the mRNA encoding the sequence of interest needs tobe estimated by, e.g.,
quantitative RT-PCR. The numbers ofrecombinants that are required can
be us high as 10 6 for rare mRNA species.
Screening based on sequence homology
The most direct method for screening a cDNA library is to use nucleic acid
probes for hybridization. This involves the following steps (Wahl and Berger 1987):
1. Aliquots of the library tobe screened (phage or plasmid transfected bacteria) are grown on master plates (or filters) from which replica filters are
made.
2. The masters are preserved while the replica filters are processed.