I"fljI Applications in Genetics
However, it is not easy to develop a PCR protocol to get a similar low amount of
PCR product for all samples, especially if one wants to avoid determining the template DNA concentration for every sample. Therefore the asymmetric PCR
approach seems to be the most practicable.
In summary, our optimized PCR protocol (asymmetric PCR with more forward
than reverse primer) allows fast and specific genotyping of the human prion protein polymorphism at codon 129 in research samples (Fig. 7).
The transmissible new variant of CJD has been known for only a few years and
the number of patients is still low. Further studies are necessary to evaluate
whether vCJD occurs in methionine homozygous individuals only, as incubation
periods may be longer in methionine/valine heterozygotes and valine homozygotes [8,13]. In this context our LightCycler assay for rapid genotyping, especially
when performed on samples obtained from the cheek mucosa (which avoids blood
drawing by a physician), can provide necessary data. In addition it might help to
predict an increased individual risk of developing transmissible forms of
Creutzfeldt-Jakob disease.
However, our findings emphasize the need for extensive validation of new
LightCycler tests. In addition, it is absolutely necessary to include controls for
every possible genotype in each run. With this in mind, a melting point analysis
using the LightCycler is an excellent alternative to other more laborious and timeconsuming techniques for the detection of single nucleotide polymorphisms in
research applications.
References
1. Windl 0, Dempster M, Estibeiro JP, Lathe R, de Silva R, Esmonde T, Will R, Springbett A,
Campbell TA, Sidle KC, Palmer MS, Collinge J (1996) Genetic basis of Creutzfeldt-Jakob disease in the United Kingdom: a systematic analysis of predisposing mutations and allelic
variation in the PRNP gene. Hum Genet 98:259-264
2. Parchi P, Giese A, Capellari S, Brown P, Schulz-Schaeffer W, Windl 0, Zerr I, Budka H, Kopp
N, Piccardo P, Poser S, Rojiani A, Streichemberger N, Julien J, Vital C, Ghetti B, Gambetti P,
Kretzschmar H (1999) Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects. Ann Neurol46:224-233
3. Masullo C, Macchi G (2001) Does PRNP gene control the clinical and pathological phenotype of human spongiform transmissible encephalopathies? Clin Neuropathol20:19-25
4. Hauw JJ, Sazdovitch V, Laplanche JL, Peoc'h K, Kopp N, Kemeny J, Privat N, DelasnerieLaupretre N, Brandel JP, Deslys JP, Dormont D, Alperovitch A (2000) Neuropathologic variants of sporadic Cieutzfeldt-Jakob disease and codon 129 of PrP gene. Neurology
54:1641-1646
5. Laplanche JL, Delasnerie-Laupretre N, Brandel JP, Chatelain J, Beaudry P, Alperovitch A,
Launay JM (1994) Molecular genetics of prion diseases in France. French Research Group on
Epidemiology of Human Spongiform Encephalopathies. Neurology 44:2347-2451
6. Deslys JP, Marce D, Dormont D (1994) Similar genetic susceptibility in iatrogenic and sporadic Creutzfeldt-Jakob disease. J Gen Virol75:23-27
7. Salvatore M, Genuardi M, Petraroli R, Masullo C, D' Alessandro M, Pocchiari M (1994) Polymorphisms of the prion protein gene in Italian patients with Creutzfeldt-Jakob disease. Hum
Genet 94:375-379
8. Ironside JW, Head MW, Bell JE, McCardle L, Will RG (2000) Laboratory diagnosis of variant
Creutzfeldt-Jakob disease. Histopathology 37:1-9
However, it is not easy to develop a PCR protocol to get a similar low amount of
PCR product for all samples, especially if one wants to avoid determining the template DNA concentration for every sample. Therefore the asymmetric PCR
approach seems to be the most practicable.
In summary, our optimized PCR protocol (asymmetric PCR with more forward
than reverse primer) allows fast and specific genotyping of the human prion protein polymorphism at codon 129 in research samples (Fig. 7).
The transmissible new variant of CJD has been known for only a few years and
the number of patients is still low. Further studies are necessary to evaluate
whether vCJD occurs in methionine homozygous individuals only, as incubation
periods may be longer in methionine/valine heterozygotes and valine homozygotes [8,13]. In this context our LightCycler assay for rapid genotyping, especially
when performed on samples obtained from the cheek mucosa (which avoids blood
drawing by a physician), can provide necessary data. In addition it might help to
predict an increased individual risk of developing transmissible forms of
Creutzfeldt-Jakob disease.
However, our findings emphasize the need for extensive validation of new
LightCycler tests. In addition, it is absolutely necessary to include controls for
every possible genotype in each run. With this in mind, a melting point analysis
using the LightCycler is an excellent alternative to other more laborious and timeconsuming techniques for the detection of single nucleotide polymorphisms in
research applications.
References
1. Windl 0, Dempster M, Estibeiro JP, Lathe R, de Silva R, Esmonde T, Will R, Springbett A,
Campbell TA, Sidle KC, Palmer MS, Collinge J (1996) Genetic basis of Creutzfeldt-Jakob disease in the United Kingdom: a systematic analysis of predisposing mutations and allelic
variation in the PRNP gene. Hum Genet 98:259-264
2. Parchi P, Giese A, Capellari S, Brown P, Schulz-Schaeffer W, Windl 0, Zerr I, Budka H, Kopp
N, Piccardo P, Poser S, Rojiani A, Streichemberger N, Julien J, Vital C, Ghetti B, Gambetti P,
Kretzschmar H (1999) Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects. Ann Neurol46:224-233
3. Masullo C, Macchi G (2001) Does PRNP gene control the clinical and pathological phenotype of human spongiform transmissible encephalopathies? Clin Neuropathol20:19-25
4. Hauw JJ, Sazdovitch V, Laplanche JL, Peoc'h K, Kopp N, Kemeny J, Privat N, DelasnerieLaupretre N, Brandel JP, Deslys JP, Dormont D, Alperovitch A (2000) Neuropathologic variants of sporadic Cieutzfeldt-Jakob disease and codon 129 of PrP gene. Neurology
54:1641-1646
5. Laplanche JL, Delasnerie-Laupretre N, Brandel JP, Chatelain J, Beaudry P, Alperovitch A,
Launay JM (1994) Molecular genetics of prion diseases in France. French Research Group on
Epidemiology of Human Spongiform Encephalopathies. Neurology 44:2347-2451
6. Deslys JP, Marce D, Dormont D (1994) Similar genetic susceptibility in iatrogenic and sporadic Creutzfeldt-Jakob disease. J Gen Virol75:23-27
7. Salvatore M, Genuardi M, Petraroli R, Masullo C, D' Alessandro M, Pocchiari M (1994) Polymorphisms of the prion protein gene in Italian patients with Creutzfeldt-Jakob disease. Hum
Genet 94:375-379
8. Ironside JW, Head MW, Bell JE, McCardle L, Will RG (2000) Laboratory diagnosis of variant
Creutzfeldt-Jakob disease. Histopathology 37:1-9