Harnessing asymmetrical substrate recognition by thermostable EndoV to achieve balanced linear amplification in multiplexed SNP typing.
Multiplexed amplification of specific DNA sequences, by PCR or by strand-displacement amplification, is an intrinsically biased process. The relative abundance of amplified DNA can be altered significantly from the original representation and, in extreme cases, allele dropout can occur. In this paper, we present a method of linear amplification of DNA that relies on the cooperative, sequence-dependent functioning of the DNA mismatch-repair enzyme endonuclease V (EndoV) from Thermotoga maritima (Tma) and Bacillus stearothermophilus (Bst) DNA polymerase. Tma EndoV can nick one strand of unmodified duplex DNA, allowing extension by Bst polymerase. By controlling the bases surrounding a mismatch and the mismatch itself, the efficiency of nicking by EndoV and extension by Bst polymerase can be controlled. The method currently allows 100-fold multiplexed amplification of target molecules to be performed isothermally, with an average change of <1.3-fold in their original representation. Because only a single primer is necessary, primer artefacts and nonspecific amplification products are minimized.
Turner
Daniel J
DJ
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA.
Pingle
Maneesh R
MR
Barany
Francis
F
D020137:Base Pair Mismatch; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D066298:In Vitro Techniques; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology
D017931:DNA Primers; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer)