Method for determining the presence of at least one allele...

Details Method for determining the presence of at least one allele... Method for determining the presence of at least one allele...

2001-02-22

2004-04-20

Souaya, Jehanne (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S023100, C536S024300, C536S024310

Reexamination Certificate

active

06723508

ABSTRACT:

This invention relates to a method for detecting the presence of at least one single allele of a deletion mutant, a PCR assay for detecting the presence of at least one GSTT1* allele and a procedure for diagnostic testing of patients to check whether they are susceptible to toxins or resistant to certain therapeutic agents or belonging to risk groups.
Human glutathione S-transferase theta (GSTT1) is an important detoxification enzyme comprising a deletion polymorphism. Approximately 20% of Caucasians are homozygous GSTT1*0/0 failing to express any GSTT1 activity. Non conjugators may have an impaired ability to metabolically eliminate toxic compounds and may therefore be at increased risk for cancer, inflammatory diseases or chemical poisoning.
Any conclusion drawn from current genotyping was limited because heterozygous (*A/0) and homozygous (*A/A) samples could yet not be discriminated. Phenotypically suggested high- and intermediate conjugators remained genotypically unexplained. The classification of all three genotypes has so far been hampered by the elucidation of the correct molecular mechanism of the GSTT1 deletion.
Thus, it is the object of this invention to provide a method for detecting the presence of at least one single allele of this deletion mutant.
This problem is solved by a method for detecting the presence of at least one single allele of a deletion mutant, wherein a PCR is performed with two primers, of which one stems from the sequence upstream of the deletion area, and the other stems from the sequence downstream of deletion area, wherein the production of the corresponding DNA fragment in a PCR is checked.
It is preferred to use this invention for detecting the presence of at least one GSTT
1
*
0
allele, wherein a combination of one primer from the enclosed sequence
1
and one primer from the enclosed sequence
2
is used and specific DNA fragments for the GSTT
1
*
0
allele are obtained by PCR.
Thereby, using the following primers showed very good results:
CAG TTG TGA GCC ACC GTA CCC
(SEQ ID NO: 21)

CGA TAG TTG CTG GCC CCC TC
(SEQ ID NO: 22)
A special purpose of this invention is to use the invention in a procedure for diagnostic testing of patients to check whether they are susceptible to toxins or resistant to certain therapeutic agents or belonging to risk groups, wherein
blood samples from the patients are obtained and genomic DNA is prepared from these blood samples
a PCR-Mapping of the obtained DNA is performed using a combination of one primer from the enclosed sequence 1 and one primer from the enclosed sequence 2 and
it is analysed whether corresponding DNA-fragments have been produced by the PCR.
In a preferred embodiment, there is an additional PCR mapping of the obtained DNA using a primer pair from within the GSTT1 gene and it is analysed whether PCR fragments of the primers according to claim
3
and/or PCR fragments of the primers from within the GSTT1 gene have been produced. In this way, all possible allele combinations (GSTT1*0/0, GSTT1*A/0 and GSTT1*A/A) can be detected.
It is preferred to use this GSTT1-genotyping assay to predict the risk for UV mediated skin damage and/or the genetic risk for skin cancer and/or the genetic risk for cancers that are associated with oxidative stress and/or damage.
According to this invention, it is further possible to obatin a quantitative PCR assay for detecting the number of active GSTT1 alleles using the ABI TaqMan® technology.
With the method according to this invention, it is preferrably possible to use the knowledge of the exact GSTT1 copy number to develop quantitative PCR based assays for detecting the number of active GSTT1 alleles using the ABI TaqMan® technology.
It is further possible according to the invention to use the knowledge of the exact numbers of GSTT1 copoies obtained according to the invention to calibrate quantitative PCR based assays for detecting the number of active GSTT1 alleles using the ABI TaqMan® technology.
The knowledge of the exact GSTT1 copy numbers obtained according to the invention can also be used to calibtrate assays for detecting the number of active GSTT1 alleles.


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