16
EIGIL KJELDSEN and STEEN K0LVRAA
labeled probe is prehybridized shortly with an excess of unlabeled repetitive DNA (Cot-I) before application to the slide. The Cot-l DNA binds to
the probes repeated sequences which in turn cannot hybridize to the chromosomal repetitive DNA leaving the unique sequences contained in the
probe single-stranded and free to hybridize to the chromosomal DNA.
Recently, a complete set of human chromosome-specific painting probes
depleted in repeated sequences have been generated (Bolzer et al. 1999).
These probes yield highly specific signals and can be hybridized without a
prehybridization step of the labeled probe.
For an efficient hybridization to take place, a number of aspects must
be considered:
The specificity of the hybridization (also known as stringency) is a crucial point. For example, if hybridization is carried out at too high a temperature, no probe annealing will take place because both probe and target
molecules remain predominantly single-stranded. On the other hand, if
the temperature is too low, the probe may anneal at many different unspecific sites on the target DNA which may not be removed in the posthybridization washing step. The appropriate temperature must be determined by experimentation although some general rules do apply.
Another way to achieve specific annealing is to alter the chemical composition of the hybridization mixture. This can be accomplished in a number of ways: (1) increasing the formamide concentration, thereby destabilizing mismatched hybrids. This has the same effect as increasing the
temperature; (2) reducing the salt concentration. This also increases
the specificity; (3) increasing the length of the probe. This increases
the specificity of the annealing process. When using YACs, cosmids or
chromosome-derived probes, the individual labeled molecules must be
between 300 and 500 bp. This size is determined by the amount of DNase
or length of nick-time when labeling the probe in nicktranslation, (4) increasing the probe concentration. This drives the reaction towards increased formation of probe-target hybrids thereby speeding up the reaction, but may also lead to non-specific background staining (in general, a
probe concentration of 1-2 ng/IJI is optimal but must be determined empirically), (5) Varying hybridization time. The time required for hybridization depends on the target sequence, i.e. highly repetitive DNA generally requires a short hybridization time (2-4 h) while low or single copy
targets require overnight hybridization, and (6) the purity of the DNA
probe.
Usually, the hybridization is performed under a sealed coverslip in a
moist chamber to minimize the evaporation of water which would otherwise alter the hybridization conditions in an uncontrolled manner.
EIGIL KJELDSEN and STEEN K0LVRAA
labeled probe is prehybridized shortly with an excess of unlabeled repetitive DNA (Cot-I) before application to the slide. The Cot-l DNA binds to
the probes repeated sequences which in turn cannot hybridize to the chromosomal repetitive DNA leaving the unique sequences contained in the
probe single-stranded and free to hybridize to the chromosomal DNA.
Recently, a complete set of human chromosome-specific painting probes
depleted in repeated sequences have been generated (Bolzer et al. 1999).
These probes yield highly specific signals and can be hybridized without a
prehybridization step of the labeled probe.
For an efficient hybridization to take place, a number of aspects must
be considered:
The specificity of the hybridization (also known as stringency) is a crucial point. For example, if hybridization is carried out at too high a temperature, no probe annealing will take place because both probe and target
molecules remain predominantly single-stranded. On the other hand, if
the temperature is too low, the probe may anneal at many different unspecific sites on the target DNA which may not be removed in the posthybridization washing step. The appropriate temperature must be determined by experimentation although some general rules do apply.
Another way to achieve specific annealing is to alter the chemical composition of the hybridization mixture. This can be accomplished in a number of ways: (1) increasing the formamide concentration, thereby destabilizing mismatched hybrids. This has the same effect as increasing the
temperature; (2) reducing the salt concentration. This also increases
the specificity; (3) increasing the length of the probe. This increases
the specificity of the annealing process. When using YACs, cosmids or
chromosome-derived probes, the individual labeled molecules must be
between 300 and 500 bp. This size is determined by the amount of DNase
or length of nick-time when labeling the probe in nicktranslation, (4) increasing the probe concentration. This drives the reaction towards increased formation of probe-target hybrids thereby speeding up the reaction, but may also lead to non-specific background staining (in general, a
probe concentration of 1-2 ng/IJI is optimal but must be determined empirically), (5) Varying hybridization time. The time required for hybridization depends on the target sequence, i.e. highly repetitive DNA generally requires a short hybridization time (2-4 h) while low or single copy
targets require overnight hybridization, and (6) the purity of the DNA
probe.
Usually, the hybridization is performed under a sealed coverslip in a
moist chamber to minimize the evaporation of water which would otherwise alter the hybridization conditions in an uncontrolled manner.