BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies
نویسندگان
چکیده
The tricarboxylate reagent benzene-1,3,5-triacetic acid (BTA) was used to attach 5'-aminated DNA primers and templates on an aminosilanized glass surface for subsequent generation of DNA colonies by in situ solid-phase amplification. We have characterized the derivatized surfaces for the chemical attachment of oligonucleotides and evaluate the properties relevant for the amplification process: surface density, thermal stability towards thermocycling, functionalization reproducibility and storage stability. The derivatization process, first developed for glass slides, was then adapted to microfabricated glass channels containing integrated fluidic connections. This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation. Innovative analytical methods for the characterization of attached DNA were developed for assessing the surface immobilized DNA content after amplification. The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm(2) from the amplification of initial single-template DNA molecules immobilized. We also demonstrate that the dsDNA colonies generated can be quantitatively processed in situ by restriction enzymes digestion. DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studies.
منابع مشابه
A novel optical DNA biosensor for detection of trace concentration of Methylene blue using Gold nano-particles and Guanine rich single strand DNA
The glass surface modification with 3-(mercaptopropyl) trimethoxysilane (MPTS), gold nano-particles (GN) and guanine rich single strand DNA (ss-DNA) was utilized as a novel and efficient platform for sensing trace concentration of methylene blue (MB) by an inexpensive spectrophotometric method. Methylene Blue (MB) can interact with the guanine base of single strand DNA and absorbed onto glass s...
متن کاملA novel optical DNA biosensor for detection of trace concentration of Methylene blue using Gold nano-particles and Guanine rich single strand DNA
The glass surface modification with 3-(mercaptopropyl) trimethoxysilane (MPTS), gold nano-particles (GN) and guanine rich single strand DNA (ss-DNA) was utilized as a novel and efficient platform for sensing trace concentration of methylene blue (MB) by an inexpensive spectrophotometric method. Methylene Blue (MB) can interact with the guanine base of single strand DNA and absorbed onto glass s...
متن کاملSolid phase DNA amplification: characterisation of primer attachment and amplification mechanisms.
Different chemical methods used to attach oligonucleotides by their 5'-end on a glass surface were tested in the framework of solid phase PCR where surface-bound instead of freely-diffusing primers are used to amplify DNA. Each method was first evaluated for its capacity to provide a high surface coverage of oligonucleotides essentially attached via a 5'-specific linkage that satisfyingly withs...
متن کاملAttachment of benzaldehyde-modified oligodeoxynucleotide probes to semicarbazide-coated glass.
Attachment of oligodeoxynucleotides (ODNs) containing benzaldehyde (BAL) groups to semicarbazide-coated glass (SC-glass) slides is described. 5'-BAL-ODNs are prepared using automated DNA synthesis and an acetal-protected BAL phosphoramidite reagent. The hydrophobic protecting group simplifies purification of BAL-ODNs by reverse phase HPLC and is easily removed using standard acid treatment. The...
متن کاملEvaluation of Genetic Variability in a Breeder Flock of Native Chicken Based on Randomly Amplified Polymorphic DNA Markers
A study was undertaken to evaluate the genetic variation in the 10th generation of a breeder flock of native breed selected for high egg and meat production in native fowls breeding station, Mazandaran, Iran. Venous blood samples were collected from 100 birds of both sexes. The RAPD-PCR technique was applied to generate a DNA fingerprint of individuals. Initially, a total of 20 ten-nucleotide a...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Nucleic Acids Research
دوره 34 شماره
صفحات -
تاریخ انتشار 2006