Ligase reaction mediated amplification method and use thereof

#This invention relates to an amplification method, particularly a reaction-medicated amplification method of ligase.

#Nucleic acid testing (NAT) has been widely applied in the clinical diagnosis of molecular genetics, immunology, oncology, microbiology and other aspects. With the rapid development of molecular biology, some new methods of nucleic acid testing are constantly emerging, wherein the detection technique of ligase dependent is one of the categories. Ligase is an enzyme to close nicks on DNA or RNA chain, which can catalyst the reaction of 3′-end hydroxyl and 5′-end phosphate group of 2 single-stranded nucleic acid to form the phosphate diester bond with the aid of energy provided by NAD+ or ATP hydrolysis. The detection technique of ligase dependent is achieved mainly by the principles of nucleic acid hybridization and its fidelity at the nick junction. In 1988, Landegren firstly invented the in vitro mutation detection technique OLA of ligase, and applied it to identify the single-base difference between the mutant alleles βS and wild type alleles βA which encode β globulin of causing Mediterranean sicklemia. In 1991, Barry extracted thermostable ligase and developed the techniques of LDR (ligase detection reaction) and LCR (ligase chain reaction). The raise of these two techniques has far-reaching significance to the development and application of the in vitro detection technique of ligase dependent, but each of them has nonspecific signal interference, poor usability and other problems. Thereafter, ligase reaction technique, combing with PCR technique, derives a series of new detection techniques, such as LDR/PCR, MLPA, PLP (padlock probe), MIP (molecular inversion probe), etc. These techniques conduct detection by achieving the “specific transformation” of the target genes by ligase reaction, and with the aid of the specific products after being amplified and transformed by PCR system. All these techniques present a relatively high detection flux, and can even achieve genotyping >10000 (MIP technique), however, they all have problems of serious nonspecific signal interference and false positive. The nonspecific signals of these techniques mainly come from: 1. nonspecific ligase reaction; 2. nonspecific PCR amplification. Influenced by the two non-specificities, the sensibility of the detection of LDR/PCR and MLPA techniques are limited; and even the nonspecific signals can be detected, the risk of false positive still exists. By adopting the method of exonuclease digesting the non-linked hybridization probes, PLR and MIP techniques eliminate the nonspecific amplification of the hybridization probes in PCR reaction to some extent; but when the detection of multiplicity is relatively high, the techniques will also be influenced by the nonspecific linking, and the presence of false positive is inevitable.

#The technical problem to be solved in this invention is: provide an amplification method that can overcome ligase reaction and nonspecific signal interference of PCR. This technique achieves the objectives of reducing reaction background, enhancing signal-to-noise ratio, avoiding false positive and others.

#FIG. 1 is a schematic view of an illustration showing the detection principle of this new type of reaction-medicated amplification method of ligase, wherein 7 is the target sequence. The target sequence is from 3′-end to 5′-end, and is divided into A, B, C and D in turn. 1 is the upstream primer tag sequence, 2 is the reversed complimentary sequence of segment A, 3 is the reversed complimentary sequence of segment B, 3′ is the reversed complimentary sequence of segment C, 4 is the detection tag sequence between segment B and C, 5 is the reversed complimentary sequence of segment D, and 6 is the downstream primer combination tag sequence.

#FIGS. 2A, 2B and 2C are all graph illustrations of the ordinary and cystic structural hybridization sequence melting curve graphs, wherein 8 are linking probes, 2 are melting curves, 9 is the linking probe 1 melting curve, 10 is the linking probe 3 melting curve, 11 is the linking probe 4 melting curve, 12 is the linking probe 2 melting curve, 13 is the linking probe 4 melting curve, and 14 is the linking probe 5 melting curve.

#FIGS. 3A and 3B are graph illustrations of the amplification curve of gradient diluting DNA and the standard curve of gradient diluting DNA, respectively; wherein 15 is the amplification curve of the gradient dilution template (sample amounts are placed in order from left to right, respectively, 1.0×107, 1.0×106, 1.0×105, 1.0×104, 1.0×103 and 1.0×102 copy), 16 is the amplification curve of negative control.

#The embodiments of this invention will be described in detail with embodiments in the following. However, as the technical personnel in this field will understand, the following embodiments are only to introduce this invention, but should not be regarded as to define the scope of this invention. Any part in the embodiments, indicated with no specific technologies or conditions, should be conducted according to the technologies or conditions described in the literatures of this field (such as in Reference: J. Same Brooke et al. Huang Yian et al (translators). The Experimental Guide of Molecular Cloning, third edition. Science press.), or product instructions. The applied reagents or instruments, indicated with no manufacturer, are all conventional products that can be purchased in the public market.

#The applied instruments in the embodiments: real-time fluorescence PCR (Rotor-gene 600, QIAGEN, Germany), ultraviolet visible spectrophotometer (ND-1000, NanoDrop, the United States), and benchtop microcentrifuge (Eppendorf, Germany). All the synthesized sequences in the embodiments of this invention are from Sangon Biotech. Ligase is AmpligaseDNA Ligase Kit (5 U/MI, 1000 U, Epicentre). The applied genomic DNA samples, all acquired by using DNeasy™ Blood Kit from Oiagen, are from peripheral blood extraction from average people, according to the extraction methods in the instructions. Peripheral blood samples are provided by Xiamen Matemal and Child Health Hospital. All the applications of samples have obtained the permissions from the parties involved or their guardians.

#Target sequence: