Comparison of allelic ratios from paired blood and paraffin-embedded normal tissue for use in a polymerase chain reaction to assess loss of heterozygosity.
BACKGROUND
One method to assess loss of heterozygosity (LOH) of various genes is the amplification of DNA from neoplastic tissue by using microsatellite markers. LOH can best be considered on a quantitative basis as a comparison of allelic ratios of neoplastic tissue to that of the normal control. We will illustrate through quantitative methods the importance of using the appropriate controls when determining allelic loss.


RESULTS
DNA extracted from 28 paired blood and formalin-fixed, paraffin-embedded normal mucosal tissue was amplified using the DP1 microsatellite marker, consisting of a variable number of CA repeats. This marker is located within the D5S346 (DP1) region on chromosome 5 and is linked to the adenomatous polyposis coli gene. Allelic ratios were calculated after scanning autoradiographs on a densitometer. Ratio values approaching 1 were observed when the two alleles were close in molecular weight, whereas ratios less than 1 were detected when the two alleles had very different molecular weights. This discrepancy was more pronounced in paraffin-embedded tissue than with blood samples.


CONCLUSIONS
For LOH amplification assays, it is best to use normal control samples that are of the same tissue source as the neoplastic sample being analyzed. When assessing LOH in neoplastic tissue, a quantitative value rather than visual assessment of the alleles should be considered. The values may be normalized by dividing the ratio of the two tumor alleles by the ratio of the two normal alleles.
Zauber
N P
NP
Department of Medicine, Saint Barnabas Medical Center, Livingston, NJ, USA.
Sabbath-Solitare
M
M
Marotta
S P
SP
McMahon
L
L
Bishop
D T
DT
D000483:Alleles; D001773:Blood Cells / Q000737:chemistry*; D002895:Chromosomes, Human, Pair 5 / Q000235:genetics; D003106:Colon / Q000737:chemistry*; D004247:DNA / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D003720:Densitometry; D005819:Genetic Markers; D006801:Humans; D007413:Intestinal Mucosa / Q000737:chemistry*; D019656:Loss of Heterozygosity; D018895:Microsatellite Repeats; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*
D005819:Genetic Markers; D004247:DNA