292
JOHANN PETER HOSSLE
Screening using
synthetic oligonucleoid probes
PCR amplified
probes using degenerate primers
order to assure specificity, hybridization reactions must be performed under the most stringent conditions possible. Stringency can be adjusted
either during hybridization or afterwards in the post -hybridization washes.
Labeling of probes can be radioactive or nonradioactive. There are many
different protocols for labeling nucleic acids with 32 P, which is the most
widely used isotope for this purpose. Since the introduction of 33 P, a low
energy ß-emitter labeling has become even more convenient. Colony
and plaque hybridization screening has also been successfully performed
using non-radioactive systems, e.g. digoxigenin (Höltke et al. 1995).
If there is not enough information available about the nucleotide sequence of
interest, it may be possible to design a synthetic oligonucleotide probe based
on partial amino acid sequence. Alternatively, one may use synthetic oligonucleotide probes to check the presence ofa particular known mutation. In
most cases where oligonucleotide probes are derived from protein sequences
itwill be necessaryto design an oligonucleotide mixture basedon codon usage
duetoredundandencyofthe geneticcode. Whensynthesizingsuch probes, the
number of different oligonucleotides (complexity) present in the mixture
should be taken in account, and should as a rule of thumb not exceed 128
(Wallace and Miyada 1987). If more complex combinations must be tested,
oligonucleotide mixtures should be hybridized as several separate pools.
For calculating the appropriate hybridization temperature (Th) the following equations are commonly used: Th = Td -5° with Ta(the temperature
at which half of the duplexes is dissociated) determined as: T d = 2° (number
of A + T nucleotides) + 4 o (number of G+C nucleotides) ( applies to duplexes
of 11 to 23 bases in 1 M Na+).
A very rapid colony hybridization screening method for oligonucleotide
probes has been developed (Paddock 1993 ), in which a special filter paper
(Whatman 541) is used rather than conventional nitrocellulose or nylon
membranes.
It should be noted that acid depurination of the DNA, which facilitates
blot transfer, should be avoided in oligonucleotide screening since oligonucleotides do not hybridize efficiently to DNA treated in this manner
(Wallace and Miyada 1987).
An approach for identification and subcloning of related cDNA sequences
(e.g. gene families) that is similar to the synthesis of complex oligonucleotide mixes for screening makes use of the polymerase chain reaction (PCR).
DNA derived from pools of cDNA clones serves as a template in PCR amplifications using degenerate primers. Primer pairs are derived from conserved amino acid sequences bracketing morediverse sequences. Amplifi-
JOHANN PETER HOSSLE
Screening using
synthetic oligonucleoid probes
PCR amplified
probes using degenerate primers
order to assure specificity, hybridization reactions must be performed under the most stringent conditions possible. Stringency can be adjusted
either during hybridization or afterwards in the post -hybridization washes.
Labeling of probes can be radioactive or nonradioactive. There are many
different protocols for labeling nucleic acids with 32 P, which is the most
widely used isotope for this purpose. Since the introduction of 33 P, a low
energy ß-emitter labeling has become even more convenient. Colony
and plaque hybridization screening has also been successfully performed
using non-radioactive systems, e.g. digoxigenin (Höltke et al. 1995).
If there is not enough information available about the nucleotide sequence of
interest, it may be possible to design a synthetic oligonucleotide probe based
on partial amino acid sequence. Alternatively, one may use synthetic oligonucleotide probes to check the presence ofa particular known mutation. In
most cases where oligonucleotide probes are derived from protein sequences
itwill be necessaryto design an oligonucleotide mixture basedon codon usage
duetoredundandencyofthe geneticcode. Whensynthesizingsuch probes, the
number of different oligonucleotides (complexity) present in the mixture
should be taken in account, and should as a rule of thumb not exceed 128
(Wallace and Miyada 1987). If more complex combinations must be tested,
oligonucleotide mixtures should be hybridized as several separate pools.
For calculating the appropriate hybridization temperature (Th) the following equations are commonly used: Th = Td -5° with Ta(the temperature
at which half of the duplexes is dissociated) determined as: T d = 2° (number
of A + T nucleotides) + 4 o (number of G+C nucleotides) ( applies to duplexes
of 11 to 23 bases in 1 M Na+).
A very rapid colony hybridization screening method for oligonucleotide
probes has been developed (Paddock 1993 ), in which a special filter paper
(Whatman 541) is used rather than conventional nitrocellulose or nylon
membranes.
It should be noted that acid depurination of the DNA, which facilitates
blot transfer, should be avoided in oligonucleotide screening since oligonucleotides do not hybridize efficiently to DNA treated in this manner
(Wallace and Miyada 1987).
An approach for identification and subcloning of related cDNA sequences
(e.g. gene families) that is similar to the synthesis of complex oligonucleotide mixes for screening makes use of the polymerase chain reaction (PCR).
DNA derived from pools of cDNA clones serves as a template in PCR amplifications using degenerate primers. Primer pairs are derived from conserved amino acid sequences bracketing morediverse sequences. Amplifi-