RQ-PCR of IG and TCR gene rearrangements can be used to quantify MRD levels by using allele-specific oligonu-cleotide (ASO) probes. Sensitivities of 1 x 10-3 to 1 x 10-5, comparable to results with the dot-blot method (Figure 31-4),55-61 are achievable with this strategy. Although initial assays used an ASO fluorescent probe to the junctional region, a more-useful approach is to use a fluorescent probe complementary to the germline IGH and TCR gene segments, in combination with an ASO primer complementary to the junctional region.56,57 The ASO primer approach theoretically results in more-sensitive MRD detection compared with use of germline primers, because no competition can occur with the amplification of similar rearrangements in normal cells. Although specific amplification can be easily distinguished from incidental nonspecific amplification, conditions with higher stringency of amplification may need to be used to overcome nonspecific amplification while maintaining the efficiency of the method.54
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