Act as both a 5′-3 that is a class of nucleic acid degrading enzymes (nucleases link large leaf endonucleases marshes known as 5 Further,)’ Biology endonuclease and exonuclease and DNA DNA recombination structure-specific DNA replication of a special structure that occur during processes, and DNA repair. Leaf endonuclease has been identified in some viruses eukaryotes, prokaryotes, and archaea. May have a plurality of FEN homologue This reduction can provide insight into the importance of these enzymes. It was found as an N-terminal domain of DNA polymerase I FEN prokaryotic enzyme, prokaryote some, seems to encode the second homologue.
It was identified by dual quality DNA is first “issued by” (referred to as name thread flap endonuclease 5 therefore “5 single-stranded flap)” In endonuclease activity of wetlands. Wetland catalyzes the hydrolysis of phosphodiester bonds at the junction of single-and double-stranded DNA. It is possible to function O to the end of the flap chain Swamp also ‘steal’ as the 5 ‘exonuclease’ DNA substrate 5′-3. Based on X-ray crystallographic data, model protein structure. Leaves end indicate that it is arched flexible created by two α-helices by through “the loop threads in the lagoon 5 ‘valve body 5 can be It has been used in the field of biotechnology, for example, single nucleotide polymorphisms Invader and TaqMan PCR assay nuclease, (SNP) analysis.
I have considered repair certain types of DNA double-strand breaks and to proceed through DNA flap, the intermediate structure. DNA flap is a forked structure consisting of the displacement of one strand and double-stranded DNA. To identify the DNA flap cleavage activity of the nuclear extracts of mammalian and established an assay using synthetic substrates, the DNA flap. In this study, it is right in the first purification DNA structure-specific nuclease of mammals. Are described herein, the flap endonuclease 1 (FEN-1), the DNA sequence, enzyme, Tan cut a flap, having the 5 ‘single-stranded end. Expected to enzyme that functions in double-strand break repair valve as resolution, FEN-1 cleavage a flap chain specific, I depend on the chain length of the flap. In addition, all the valve body is required for cleavage of valve effective. The lack substrate in one direction, it is torn from the fan 1. In addition, the branch structure of the others, including the Holiday junction, it is not cleaved by FEN-1. In addition to endonuclease activity, the fan 1 has a 5′-3 ‘exonuclease activity specific for double-stranded DNA. End of the fan 1 – exonuclease activity and are described in the context of the repair DNA replication and recombination.
Such as 1993 Lyamichev. The ability of the DNA polymerase of single-stranded DNA with a base sequence which can not be cleaved recognize how the structure is specific is described. Based on the bifurcated structure formed solely 5 ‘exonuclease activity to remove excess nucleotides in the DNA during replication is not based on any sequence motifs. The (isolated FEN from fulgidus and archeae Pyrococcus Julio) as shown in the wing of the heat-resistant endonuclease, in addition to shows similar characteristics, and the detailed analysis, the structure of the specific cleavage mismatch array such as Lyamichev then Because it showed that to be very sensitive, polymorphism DNA that enables the use of endonuclease activity for high sensitivity detection. In this preliminary study, these enzymes, suggesting that without the need for genotyping reaction or PCR thermal cycling may be used in particular to interrogate the SNP.
We describe the different approaches to the invader ® assay based on the application and its current SNP genotyping in detail below. In addition, we can discuss the new platforms that take advantage of analysis Invaders ®, to provide alternative approaches to trade the available analysis format currently.
Format described allows the genotyping of the SNP very specific, but this requires a significant amount of target molecules to generate a detectable fluorescent signal within a reasonable time. Therefore, this assay format can be Invader reaction, requiring the first PCR amplification of the target region. Furthermore, each of which must be labeled with a quencher molecule and a fluorophore, assays are required (one for each of the SNP alleles) allele-specific probes with two different. This increases the cost of research, suitable for this format for high-throughput genotyping projects and fairly large.
Invader ® assay using flap endonuclease a specific structure (FEN) a three-dimensional complex formed by hybridization of the allele-specific overlapping oligonucleotides to target DNA containing (SNP) site single nucleotide polymorphism to be cut and. In addition to the results of the target molecules in the cleavage of oligonucleotide cleavase, heat resistance by FEN, annealing of oligonucleotide SNP allele. Cleavage can be detected by several different approaches. Most commonly, cleavage product to produce a secondary cleavage reaction on (FRET) cassette fluorescence resonance energy transfer that emits fluorescence signal. Alternatively, cutting may be by the use of fluorescence polarization (FP) probes, or directly detected by mass spectrometry. Invasive cleavage reaction is highly specific, it is possible to have a low error rate, and detects the amount of target zeptomol DNA. Tests have been used to examine the one SNP in one sample per reaction traditionally, the strip efficient and accurate this fits genotyping high-throughput SNP and multiplexing chip or new approach It has been tested in order to perform the assay.