Universal or whole genome amplification by polymerase chain reaction (PCR) is a rapid and efficient method to generate fragments representing the target sequence, as well as to increase a limited amount of template. One of the most common PCR protocols for total genome amplification is the interspersed repetitive sequence-PCR (IRS-PCR) in which primers specific for human repeat-rich regions are used to generate PCR products between adjacent repeated sequences (1). However, although IRS-PCR across regions such as Alu families of human repeat has been demonstrated to be useful, the nonuniform distribution of repeat-rich region within the human genome has been a limitation. Alternative strategies have been proposed. In the primer-extension preamplification (PEP), multiple rounds of extensions with Taq DNA polymerase and a random mixture of 15-base oligonucleotides as primers produce multiple copies of the template present in the sample (2–5). In a more demanding protocol, called linker adaptor-PCR, RsaI restricted genomic DNA fragments are ligated to SmaI-cut pUC plasmid. Subsequently, the inserts are amplified by PCR using the universal M13/pUC sequencing and reverse sequencing primers and then released by EcoRI digestion (6). The tagged random primer PCR (T-PCR) is a two-step PCR strategy which consists of a pool of all possible 3'-sequences for binding to the target DNA and a constant 5'-region for the detection of incorporated primers (7). Recently, degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) was developed to allow random amplification of DNA from any source (8–10). DOP-PCR uses a partially degenerate sequence in a PCR protocol with two different annealing temperatures. It has been successfully applied for amplifying entire genomes such as human, mouse, and fruit fly, as well as isolated human chromosomes and cosmids (11). The technique has also been used to prepare whole chromosome paint probes (11,12) for micro-FISH assays (13–15), comparative genomic hybridization (16), to increase the amount of sample for genotyping (17), and genomic fingerprinting (18). The DOP-PCR primer consists of three regions. The 5'-end carries a recognition sequence for XhoI (C•TCGAG), a restriction endonuclease that cuts rarely within the human genome. This sequence can be used for cloning, if desired. The sequence is then followed by a middle portion containing six nucleotides of degenerate sequence (NNNNNN, where N = A, C, G, or T in approximately equal proportions) and a 3'-end sequence containing six specific bases (ATGTGG) which primes the reaction approximately every 4 kb (8,9). The principle of the technique is that at a sufficiently low annealing temperature only the six specific nucleotides included in the 3'-end of the degenerate oligonucleotide will anneal to the genomic strand allowing the primer to initiate PCR. The PCR fragments are then generated which contain the full length of the oligoprimer at one end and its complementary sequence at the other end. Subsequently, the temperature is increased to the level required for the full length of the degenerate primer to anneal. For additional details, we direct the reader to the original papers (8,9). We have adapted the DOP-PCR technique to a three-microchip format (19). DOP-PCR amplified genomic DNA produced in a first silicon-glass chip is transferred to a second chip for a locus-specific, multiplex PCR of the dystrophin gene exons in order to detect deletions causing Duchenne/Becker muscular dystrophy (DMD/BMD). Amplicons from the multiplex-PCR are then analyzed by electrophoresis in a third microchip. The analytical performance of the microchip capillary electrophoresis (MCE) is also compared to conventional capillary electrophoresis (CE).
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|Titolo:||DOP-PCR amplification of whole genomic DNA and microchip-based capillary electrophoresis.|
|Data di pubblicazione:||2001|
|Appartiene alla tipologia:||02a Capitolo o Articolo|