Essay About Rapid Cycle Real-Time And Standard Real-Time Pcr Method

Rapid, Long-Range Molecular Haplotyping of Thiopurine S-Methyltransferase
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Rapid, Long-Range Molecular Haplotyping of
Thiopurine S-methyltransferase
(TPMT) *3A, *3B, and *3C
Nicolas von Ahsen,* Victor W. Armstrong, and Michael Oellerich
Background: Haplotyping is an important technique in
molecular diagnostics because haplotypes are often
more predictive for individual phenotypes than are the
underlying single-nucleotide polymorphisms (SNPs).
Until recently, methods for haplotyping SNPs separated
by kilobase distances were laborious and not applicable
to high-throughput screening. In the case of thiopurine
S-methyltransferase (TPMT), differentiating among
TPMT*3A, *3B, and *3C alleles is sometimes necessary
for predictive genotyping.
Methods: The genomic region including the two SNPs
that define TPMT*3A, *3B, and *3C alleles was amplified
by long-range PCR. The resulting PCR product was
circularized by ligation and haplotyped by allele-specific
amplification PCR followed by product identification
with hybridization probes.
Results: Critical points were the long-range PCR conditions,
including choice of buffer and primers, optimization
of the ligation reaction, and selection of primers
that allowed for strict allele-specific amplification in the
second-round PCR. Different underlying TPMT haplotypes
could then be differentiated. Results from the
haplotyping method were in full agreement with those
from our standard real-time PCR method: TPMT*1/*3A
(n 20); TPMT*1/*3C (n 4); TPMT*1/*1 (n 6); and
TPMT*3A/*3A (n 6). One TPMT*1/*3A sample failed
to amplify, and no whole blood was available for repeat
DNA isolation.
Conclusions: This method for rapid cycle real-time,
allele-specific amplification PCR-assisted long-range
haplotyping has general application for the haplotyping
of distant SNPs. The procedure is simpler and more
rapid than previous methods. With respect to TPMT,
haplotyping has the potential to discriminate the genotypes
TPMT*1/*3A (intermediate metabolizer) and
TPMT*3B/*3C (poor metabolizer).
© 2004 American Association for Clinical Chemistry
One of the established targets for pharmacogenetic analyses
is thiopurine S-methyltransferase (TPMT;1 EC
2.1.1.67). Although the physiologic function of the enzyme
is still unknown, TPMT is involved in the metabolism
of the thiopurine drugs 6-mercaptopurine, 6-thioguanine,
and azathioprine. Thiopurine drugs are in common
use for the treatment of acute leukemia and autoimmune
disorders such as chronic inflammatory bowel disease,
and for