Abstract
Background: Several microdevices have been developed to perform only a single step of a genotyping process, such as PCR or detection by probe hybridization. Here, we describe a Lab-on-Chip (LoC) platform integrating a PCR amplification microreactor with a customable microarray for the detection of sequence variations on human genomic DNA. Methods: Preliminary work was focused on developing the single analytical steps including PCR and labeling strategies of the amplified product by conventional reference systems. The optimized protocols included a 1:4 forward:reverse primer ratio for asymmetric PCR, and Cy5-dCTP multiple incorporation for the generation of a labeled PCR product to be hybridized to complementary probes bound to the chip surface. Results: Final conditions were applied to the fully integrated LoC platform for the detection of the IVSI-110 G>A mutation in the human β-globin (HBB) gene associated with β-thalassemia, used as a model of genetic application, allowing for correct genotyping of 25 samples that were heterozygous, homozygous or wild-type for this mutation. Conclusions: The overall results show that the present platform is very promising for rapid identification of DNA sequence variations in an integrated, cost effective and convenient silicon chip format.
| Original language | English |
|---|---|
| Pages (from-to) | 329-336 |
| Number of pages | 8 |
| Journal | Clinical Chemistry and Laboratory Medicine |
| Volume | 48 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - Mar 1 2010 |
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Keywords
- Lab-on-Chip
- Microarray
- Microdevice
- Mutation detection
ASJC Scopus subject areas
- Clinical Biochemistry
- Biochemistry, medical
Cite this
Integrated PCR amplification and detection processes on a Lab-on-Chip platform : A new advanced solution for molecular diagnostics. / Foglieni, Barbara; Brisci, Angela; Biagio, Floriana San; Pietro, Patrizia Di; Petralia, Salvatore; Conoci, Sabrina; Ferrari, Maurizio; Cremonesi, Laura.
In: Clinical Chemistry and Laboratory Medicine, Vol. 48, No. 3, 01.03.2010, p. 329-336.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Integrated PCR amplification and detection processes on a Lab-on-Chip platform
T2 - A new advanced solution for molecular diagnostics
AU - Foglieni, Barbara
AU - Brisci, Angela
AU - Biagio, Floriana San
AU - Pietro, Patrizia Di
AU - Petralia, Salvatore
AU - Conoci, Sabrina
AU - Ferrari, Maurizio
AU - Cremonesi, Laura
PY - 2010/3/1
Y1 - 2010/3/1
N2 - Background: Several microdevices have been developed to perform only a single step of a genotyping process, such as PCR or detection by probe hybridization. Here, we describe a Lab-on-Chip (LoC) platform integrating a PCR amplification microreactor with a customable microarray for the detection of sequence variations on human genomic DNA. Methods: Preliminary work was focused on developing the single analytical steps including PCR and labeling strategies of the amplified product by conventional reference systems. The optimized protocols included a 1:4 forward:reverse primer ratio for asymmetric PCR, and Cy5-dCTP multiple incorporation for the generation of a labeled PCR product to be hybridized to complementary probes bound to the chip surface. Results: Final conditions were applied to the fully integrated LoC platform for the detection of the IVSI-110 G>A mutation in the human β-globin (HBB) gene associated with β-thalassemia, used as a model of genetic application, allowing for correct genotyping of 25 samples that were heterozygous, homozygous or wild-type for this mutation. Conclusions: The overall results show that the present platform is very promising for rapid identification of DNA sequence variations in an integrated, cost effective and convenient silicon chip format.
AB - Background: Several microdevices have been developed to perform only a single step of a genotyping process, such as PCR or detection by probe hybridization. Here, we describe a Lab-on-Chip (LoC) platform integrating a PCR amplification microreactor with a customable microarray for the detection of sequence variations on human genomic DNA. Methods: Preliminary work was focused on developing the single analytical steps including PCR and labeling strategies of the amplified product by conventional reference systems. The optimized protocols included a 1:4 forward:reverse primer ratio for asymmetric PCR, and Cy5-dCTP multiple incorporation for the generation of a labeled PCR product to be hybridized to complementary probes bound to the chip surface. Results: Final conditions were applied to the fully integrated LoC platform for the detection of the IVSI-110 G>A mutation in the human β-globin (HBB) gene associated with β-thalassemia, used as a model of genetic application, allowing for correct genotyping of 25 samples that were heterozygous, homozygous or wild-type for this mutation. Conclusions: The overall results show that the present platform is very promising for rapid identification of DNA sequence variations in an integrated, cost effective and convenient silicon chip format.
KW - Lab-on-Chip
KW - Microarray
KW - Microdevice
KW - Mutation detection
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UR - http://www.scopus.com/inward/citedby.url?scp=77249156977&partnerID=8YFLogxK
U2 - 10.1515/CCLM.2010.063
DO - 10.1515/CCLM.2010.063
M3 - Article
C2 - 20020819
AN - SCOPUS:77249156977
VL - 48
SP - 329
EP - 336
JO - Clinical Chemistry and Laboratory Medicine
JF - Clinical Chemistry and Laboratory Medicine
SN - 1434-6621
IS - 3
ER -