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BURT G. FEUERSTEIN et al.
material will not be discerned. Thus, balanced translocations or inversions
cannot be detected. In addition, since the technique relies on relative
changes in DNA copy number, alterations in ploidy will not be sensed.
Thus, a perfectly tetraploid sample cannot be distinguished from a perfectly diploid or triploid sample. Furthermore, since the resolution of the
technique is 5-10 megabases, the method cannot identify mutations or
small deletions or duplications.
Outline
The basics of the protocol for a CGH experiment are diagrammed in Fig. 1.
Subprotocol 1
Genomic DNA preparation from microdissected paraffin sections
Since CGH evaluates all DNA from a tissue, it is important to isolate DNA
from a section of tissue that has disease. If DNA from normal tissue is
included in the test sample, the sensitivity of the assay will be correspondingly reduced. After the appropriate tissue is identified, test DNA is isolated and labeled. For experiments on human material, reference DNA is
most often prepared from peripheral blood, although DNA from any normal (Le. nonneoplastic) tissue can be used.
The original descriptions of CGH specify isolation of large molecular
weight DNA from frozen tissue. The original extraction protocol included
steps to isolate and lyse nuclei, digest protein, and purify DNA with phenol/chloroform/isoamyl alcohol extractions and ethanol precipitations
(2,4).
More recently, successful CGH protocols have been developed for degenerate oligonucleotide primer polymerase chain reaction (DOP-PCR)
amplified and unamplified DNA isolated from fresh and from fixed tissues
preserved in paraffin (5-7). We describe here a recent protocol to microdissect, isolate, amplify, and label DNA from small samples that are either
fresh or fixed and preserved in paraffin. We also include a more traditional
direct labeling method. It is important to remember that the reference and
test DNAs used together in an experiment should be prepared in a similar
fashion. Thus, if the test DNA is prepared using DOP-PCR, so should the
reference DNA.
BURT G. FEUERSTEIN et al.
material will not be discerned. Thus, balanced translocations or inversions
cannot be detected. In addition, since the technique relies on relative
changes in DNA copy number, alterations in ploidy will not be sensed.
Thus, a perfectly tetraploid sample cannot be distinguished from a perfectly diploid or triploid sample. Furthermore, since the resolution of the
technique is 5-10 megabases, the method cannot identify mutations or
small deletions or duplications.
Outline
The basics of the protocol for a CGH experiment are diagrammed in Fig. 1.
Subprotocol 1
Genomic DNA preparation from microdissected paraffin sections
Since CGH evaluates all DNA from a tissue, it is important to isolate DNA
from a section of tissue that has disease. If DNA from normal tissue is
included in the test sample, the sensitivity of the assay will be correspondingly reduced. After the appropriate tissue is identified, test DNA is isolated and labeled. For experiments on human material, reference DNA is
most often prepared from peripheral blood, although DNA from any normal (Le. nonneoplastic) tissue can be used.
The original descriptions of CGH specify isolation of large molecular
weight DNA from frozen tissue. The original extraction protocol included
steps to isolate and lyse nuclei, digest protein, and purify DNA with phenol/chloroform/isoamyl alcohol extractions and ethanol precipitations
(2,4).
More recently, successful CGH protocols have been developed for degenerate oligonucleotide primer polymerase chain reaction (DOP-PCR)
amplified and unamplified DNA isolated from fresh and from fixed tissues
preserved in paraffin (5-7). We describe here a recent protocol to microdissect, isolate, amplify, and label DNA from small samples that are either
fresh or fixed and preserved in paraffin. We also include a more traditional
direct labeling method. It is important to remember that the reference and
test DNAs used together in an experiment should be prepared in a similar
fashion. Thus, if the test DNA is prepared using DOP-PCR, so should the
reference DNA.