阅读说明：本技术 Pna在检测dna裂解片段中的应用 (Application of PNA in detection of DNA cleavage fragments ) 是由 叶盛 李思慧 朱晓玲 徐涛 梁兴国 方志俊 申志发 小宫山真 于 2020-08-28 设计创作，主要内容包括：本发明公开了一种PNA在检测DNA裂解片段中的应用,以及相应的检测DNA裂解片段的方法和引物组。本发明通过基于修饰的PNA钳制PCR,来选择性扩增裂解后的DNA,而未被裂解的模板DNA则被PNA抑制,根据PCR结果即可定性或定量的分析酶促反应,可应用于监测酶活性、检测循环肿瘤DNA和循环胎儿DNA等。(The invention discloses application of PNA in detection of DNA cleavage fragments, and a corresponding method and a corresponding primer group for detecting the DNA cleavage fragments. The invention selectively amplifies the cracked DNA by clamping PCR based on the modified PNA, but the uncleaved template DNA is inhibited by the PNA, and the enzymatic reaction can be qualitatively or quantitatively analyzed according to the PCR result, and the invention can be applied to monitoring the enzymatic activity, detecting the circulating tumor DNA, circulating fetal DNA and the like.)

The use of PNA in the detection of DNA cleavage fragments.

2. A method for qualitatively detecting DNA cleavage fragments, comprising the steps of:

(1) designing sequences of a primer group and a PNA probe group, wherein the nucleotide sequences of the primer group and the PNA probe group are partially overlapped, and the overlapped part is complementary with an end nucleic acid sequence of a DNA cleavage fragment to be detected; the number of bases of the overlapping sequence of the PNA probe and the amplification primer is 9 to 11;

(2) and (3) taking a sample to be detected as a template, adding a primer group and a PNA probe group to perform PCR reaction, and then checking the result through electrophoresis, wherein a nucleic acid band can be observed to indicate that the DNA cracking fragment to be detected exists.

3. The method for qualitatively detecting DNA cleavage fragments according to claim 2, characterized in that: the primer of the primer group is a single-base mismatch primer, and a mismatch site is positioned in the middle of an overlapping sequence of the PNA probe and the nucleic acid amplification primer.

4. A method for detecting DNA cleavage fragments in real time, comprising the steps of:

(1) designing a primer group, a PNA probe group and a TaqMan MGB probe sequence, wherein the nucleotide sequences of the primer group and the PNA probe group are partially overlapped, and the overlapped part comprises an end nucleic acid sequence of a DNA cracking fragment to be detected;

(2) and (3) taking a sample to be detected as a template, adding a primer group, a PNA probe group and a TaqMan MGB probe to perform fluorescent quantitative PCR, performing quantitative analysis by a standard curve method, and analyzing an amplification result.

5. The method for detecting a DNA cleavage fragment in real time according to claim 4, wherein: the primer of the primer group is a single-base mismatch primer, and a mismatch site is positioned in the middle of an overlapping sequence of the PNA probe and the nucleic acid amplification primer.

6. A group of primer and PNA probe combination for qualitatively detecting SRY gene cleavage fragment comprises:

SRY-PNA-F：H2N-AAAGCTGTAACTCTA-CONH2

SRY-PF1m：CTGTAAATCTAAGTATCAGTGTG

SRY-PNA-R：H2N-TCCCAGCTGCTTGCT-CONH2

SRY-PR1m：AGCTGCATGCTGATCTCTGAG。

7. a group of primers and PNA, TaqMan probe combination for real-time detection of SRY gene cleavage fragment comprises:

SRY-PNA-F：H2N-AAAGCTGTAACTCTA-CONH2

SRY-PF1m：CTGTAAATCTAAGTATCAGTGTG

SRY-PNA-R：H2N-TCCCAGCTGCTTGCT-CONH2

SRY-PR1m：AGCTGCATGCTGATCTCTGAG

TaqMan-MGB-SRY：AAGCGACCCATGAA。

8. a group of primer and PNA probe combination for qualitatively detecting Maspin gene cleavage fragment comprises:

Maspin-PNA-F：H2N-CCAACGTGTCTGAGA-Lys-CONH2

Maspin-PF1m：GTGTCAGAGAAATTTGTAGTGTTAC

Maspin-PNA-R：H2N-CATACGTACAGACAT-Lys-CONH2

Maspin-PR1m：GTACAAACATGCGTACGGC。

9. a group of primers for detecting Maspin gene cleavage fragments in real time and PNA and TaqMan probe combination comprise:

Maspin-PNA-F：H2N-CCAACGTGTCTGAGA-Lys-CONH2

Maspin-PF1m：GTGTCAGAGAAATTTGTAGTGTTAC

Maspin-PNA-R：H2N-CATACGTACAGACAT-Lys-CONH2

Maspin-PR1m：GTACAAACATGCGTACGGC

TaqMan-MGB-Maspin：CGAATATTTCACCTTCC。

Technical Field

The invention belongs to the field of nucleic acid detection, and particularly relates to application of PNA in detection of DNA cleavage fragments, and a corresponding detection method.

Background

Enzymes (both natural and artificial) are one of the most valuable tools in biochemistry and biotechnology. Currently, among 3600 known restriction enzymes, more than 300 enzymes have more than 250 different specificities, which are used in laboratories, hospitals and industries to cleave a variety of DNA substrates for different purposes. And the search for new enzymes remains one of the problems. One of the common uses of restriction enzymes is the site-selective cleavage of double-stranded DNA to obtain useful fragments. However, it is not easy to analyze the efficiency of the enzymatic reaction, especially when a large amount of background DNA is present and the concentration of DNA substrate is very low, the cleaved DNA is interfered with by the background DNA and is very difficult to detect.

Peptide Nucleic Acids (PNAs) are DNA analogs with a polypeptide-like backbone formed by linking N (2-aminoethyl) -glycine to a nucleobase via a methylene carbonyl group. PNAs can specifically hybridize to DNA or RNA to form stable complexes. PNA has 3 features: 1.PNA binding ability to DNA is superior to DNA binding ability; 2. the stability of the DNA/PNA double strand is higher than that of the DNA/DNA double strand; 3. PNAs readily recognize single base mismatched DNA or RNA binding. Since the matched PNA has a higher affinity for the target site, there is an advantage over primer binding. Since Taq polymerase cannot extend PNA, this binding effect compromises the amplification reaction, which is PCR clamping. When the mutated sequence is present, the PCR primers will bind more than the PNA, thereby generating amplicons. Therefore, PNA-PCR is mainly used for the detection of point-mutated genes, which can bind to non-mutated genes, prevent primers from binding to non-mutated genes, and cannot bind to mutated genes, while primers can bind to mutated genes, thereby amplifying the mutated genes. PCR clamping can also be performed by interfering with the extension of the primers, wherein the PNA is located at a distance from the PCR primers or adjacent to one of the PCR primers. However, this PCR clamp cannot be used to detect cleaved DNA.

Disclosure of Invention

1. The invention aims to provide a novel method.

The object of the present invention is to provide the use of PNA for the detection of DNA cleavage fragments, based on which cleaved DNA can be amplified while uncleaved template DNA is suppressed, thereby enabling the detection of cleaved DNA.

2. The technical scheme adopted by the invention is disclosed.

The invention discloses application of PNA in detection of DNA cleavage fragments.

The invention discloses a method for qualitatively detecting DNA cracking fragments, which comprises the following steps:

(1) designing sequences of a primer group and a PNA probe group, wherein the nucleotide sequences of the primer group and the PNA probe group are partially overlapped, and the overlapped part comprises an end nucleic acid sequence of a DNA cleavage fragment to be detected;

(2) and (3) taking a sample to be detected as a template, adding a primer group and a PNA probe group to perform PCR reaction, and then checking the result through electrophoresis, wherein a nucleic acid band can be observed to indicate that the DNA cracking fragment to be detected exists.

Preferably, the primer of the primer set is a single base mismatch primer, and the mismatch site is located in the middle of the overlapping sequence of the PNA probe and the nucleic acid amplification primer.

The invention also discloses a method for detecting the DNA cracking fragment in real time, which comprises the following steps:

(1) designing a primer group, a PNA probe group and a TaqMan MGB probe sequence, wherein the nucleotide sequences of the primer group and the PNA probe group are partially overlapped, and the overlapped part comprises an end nucleic acid sequence of a DNA cracking fragment to be detected;

(2) and (3) taking a sample to be detected as a template, adding a primer group, a PNA probe group and a TaqMan MGB probe to perform fluorescent quantitative PCR, performing quantitative analysis by a standard curve method, and checking an amplification result.

Preferably, the primer of the primer set is a single base mismatch primer, and the mismatch site is located in the middle of the overlapping sequence of the PNA probe and the nucleic acid amplification primer.

The invention discloses a primer and PNA probe combination for qualitatively detecting SRY gene cracking fragments, which comprises the following components:

SRY-PNA-F：H2N-AAAGCTGTAACTCTA-CONH2

SRY-PF1m：CTGTAAATCTAAGTATCAGTGTG

SRY-PNA-R：H2N-TCCCAGCTGCTTGCT-CONH2

SRY-PR1m：AGCTGCATGCTGATCTCTGAG。

the invention discloses a group of primers, PNA probe and TaqMan probe combination for detecting SRY gene cracking fragment in real time, which comprises the following components:

SRY-PNA-F：H2N-AAAGCTGTAACTCTA-CONH2

SRY-PF1m：CTGTAAATCTAAGTATCAGTGTG

SRY-PNA-R：H2N-TCCCAGCTGCTTGCT-CONH2

SRY-PR1m：AGCTGCATGCTGATCTCTGAG

TaqMan-MGB-SRY：AAGCGACCCATGAA。

the invention discloses a primer and PNA probe combination for qualitatively detecting Maspin gene cracking fragments, which comprises the following components:

Maspin-PNA-F：H2N-CCAACGTGTCTGAGA-Lys-CONH2

Maspin-PF1m：GTGTCAGAGAAATTTGTAGTGTTAC

Maspin-PNA-R：H2N-CATACGTACAGACAT-Lys-CONH2

Maspin-PR1m：GTACAAACATGCGTACGGC。

the invention discloses a group of primers, a PNA probe and a TaqMan probe combination for detecting Maspin gene cracking fragments in real time, which comprises the following components:

Maspin-PNA-F：H2N-CCAACGTGTCTGAGA-Lys-CONH2

Maspin-PF1m：GTGTCAGAGAAATTTGTAGTGTTAC

Maspin-PNA-R：H2N-CATACGTACAGACAT-Lys-CONH2

Maspin-PR1m：GTACAAACATGCGTACGGC

TaqMan-MGB-Maspin：CGAATATTTCACCTTCC。

3. the technical effect produced by the invention.

(1) The invention selectively amplifies the cracked DNA by clamping PCR based on the modified PNA, but the uncleaved template DNA is inhibited by the PNA, and the enzymatic reaction can be qualitatively or quantitatively analyzed according to the PCR result. Can be used for monitoring enzyme activity, circulating tumor DNA, circulating fetal DNA and the like.

Drawings

FIG. 1 is a real-time PCR amplification of an enzymatic digest in the presence of a TaqMan-MGB-SRY probe in example 1. The samples used in curves 1-4 (1 ng undigested DNA on the left and 1ng digested DNA on the right) correspond to the samples used in lanes 1-4 in FIG. 2. Curve 1, digested DNA; curve 2, digested DNA + PNA; curve 3, uncut DNA; curve 4, uncut DNA + PNA.

FIG. 2 is an agarose gel electrophoresis analysis of PGR amplification in the absence (one) or presence (+) PNA (SRY-PNA-F and SRY-PNA-R) in example 1. Lane 1, digested DNA; lane 2, digested DNA + PNA; lane 3, uncleaved DNA; lane 4, unadigested DNA + PNA.

FIG. 3 is a real-time PCR amplification of enzymatic digests in the presence of TaqMan-MGB-Maspin probe in example 2. Left to right curve starting from the first appearance of the fluorescence signal: uncut DNA, cut DNA + PNA, uncut DNA + PNA.

FIG. 4 is an electrophoretic analysis of PCR amplification of an enzyme digest made by the enzyme HpyCH4 in the absence (- -) or presence (+) PNA in example 2. NTC, no template control; lane 1, maternal DNA; lane 2, maternal DNA + PNA; lane 3, placental DNA; lane 4, placental DNA + PNA.

FIG. 5 shows the sensitivity of PCR amplification with restriction enzyme in the presence of PNA (Maspin-PNA-F and Maspin-PNA-R) in example 3. Percentage of digested DNA from left to right curve: 100%, 10%, 5%, 1%, 0%.

FIG. 6 is a PNA-based real-time PCR assay for enzymatic digestion of HpyCH4 at various concentrations. Enzyme concentration in the reaction (left to right curve): 0 units (no PNA), 0.5 units, 0.2 units, 0.1 units, 0.05 units and 0 units. Reaction conditions are as follows: at 37 ℃ for 10 minutes.

FIG. 7 is a PNA based real-time PCR analysis of the enzymatic reaction time course. Left to right curve: the DNA digestion times were 8 hours (100% digested DNA), 2 hours, 1 hour, 30 minutes, 10 minutes and 5 minutes, respectively.

The left to right confirmation of the curves in the figures is from left to right starting from the first appearance of the fluorescence signal.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The present invention is further illustrated by the following specific examples, which are not intended to be limiting. The experimental methods and reagents of the formulations not specified in the examples are all prepared according to conventional conditions such as Sambrook et al, molecular cloning: conditions described in the test Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or conditions suggested by the manufacturer.

Example 1

1. Design and Synthesis of primers

Table 1, primers, TaqMan MGB probes and PNA used in the present invention.

The underlined sequence of the primers is identical to the underlined sequence in the corresponding PNA except for a single base substitution at base 6 or 7 of the primers for the SRY and Maspin genes. The positions of mismatch are shown in bold italics. The restriction is performed before the underlined sequence (i.e., the overlapping sequence).

2. Sample Collection and DNA preparation

Pregnant women who had been seen a doctor in obstetrics and gynecology in Weiersi's Hospital, hong Kong were recruited. Pregnant woman peripheral blood samples (12ml EDTA) and placenta samples were collected before and after caesarean section, respectively.

Blood samples were centrifuged at 1600 μ g for 10 min at 4 ℃ and then plasma fractions at 16,000 μ g for 10 min. The peripheral blood cell fraction was re-centrifuged to 5,000 μ g and any residual plasma was removed. DNA was extracted from peripheral blood cells using the Nucleon blood DNA extraction kit (GE Healthcare, Little Chalfont, uk) and from placental tissue using the QIAamp tissue kit (Qiagen, Hilden, germany). DNA was extracted from 0.8ml plasma using the QlAamp blood kit (Qiagen) according to the manufacturer's blood and body fluid protocol.

3. Enzymatic reaction

The enzymatic reaction with restriction nuclease (Alu1) was carried out on NEB buffer 2. The reaction mixture of DNA and restriction nuclease was incubated at 37 ℃ for a predetermined time and then rapidly heated to 65 ℃ for 20 minutes to terminate the enzyme reaction.

4. Real-time PCR

Real-time PCR analysis using TaqMan probes was performed on an ABI PRISM 7300 instrument (Applied Biosystems). TaqMan MGB probes (Applied Biosystems) for SRY genes were labeled with a reporter dye 6-FAM (carboxyfluorescein) at the 5 'end and a Minor Groove Binder (MGB) at the 3' end. The primers used in the present invention are listed in table 1. Primers were purchased from Sigma-Prooligo (Singapore) and Molecular information Laboratory (Korea). Extracted DNA was amplified using TaqMan Universal PCR Mastermix (Applied Biosystems) in a 50. mu.L reaction volume. All probes were used at a concentration of 50nmol/L and primers at a concentration of 250 nmol/L. Under PCR cycling conditions, 50ng of DNA template was used per 25. mu.L PCR reaction: 5 minutes at 95 ℃; 30 cycles of 95 ℃ for 30 seconds, 55 ℃ for 30 seconds and 72 ℃ for 1 minute; 7 minutes at 72 ℃. Analysis was repeated at least twice for each sample. The results were analyzed using sequence detection software version 2.1 (Applied Biosystems) according to the manufacturer's instructions. The results of the fluorescent quantitative PCR are shown in FIG. 1.

5. Gel electrophoresis

After the PCR reaction, 2-5. mu.L of the mixture was subjected to 2% agarose gel electrophoresis and analyzed using GDS-8000 Bioimaging system. The results are shown in FIG. 2. As can be seen from FIG. 2, the DNA in lanes 1 and 2 was completely digested by Alu1, resulting in short fragments. The DNA in lanes 3 and 4 remained intact and was not digested by Alu1 enzyme.

And (4) analyzing results: gel-based detection of PCR amplification the effect of PNA on PCR amplification was visualized. As shown in FIG. 2, both cleaved and uncleaved templates were efficiently amplified under the specified conditions in the absence of PNA (lanes 1 and 3). However, in the presence of PNA, PCR amplification of the uncut DNA is completely blocked, while the cut DNA is selectively amplified.

Real-time amplification detection is a more preferred protocol because of the high sensitivity and the quantitative and homogeneous assay. As shown in fig. 1, for real-time PNA-based assays, PNA substantially blocked amplification of uncleaved DNA: in the presence of PNA, no fluorescence signal was detected (Curve 4). PCR amplification of the digested DNA was not affected by PNA.

Example 2

This example was substantially identical to example 1, and the Maspin gene was digested with HpyCH4 enzyme, and the results are shown in FIGS. 3 and 4. Enzymatic reaction: a20. mu.L volume of 100ng DNA in pH 8.0 NEB buffer 1 was digested with 1 unit of HpyCH4 for 2 hours at 37 ℃. PCR cycling conditions: 95 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 30 s; 32 cycles.

The results of FIGS. 1-4 demonstrate that PCR amplification of uncleaved DNA can be completely inhibited using PNA, and that amplification of digested DNA fragments is not affected. To further evaluate the effect of PNA, Ct (the Ct difference between the absence and presence of PNA) was used to describe PCR inhibition. The larger the Ct, the higher the efficiency of PCR inhibition. The Ct was 8 in the Alu1 system and 15 in the HpyCH4 system, indicating that only a small trace of uncut DNA could escape inhibition by PNA during PCR.

Example 3

PNA-mediated PCR was tested for sensitivity to low levels of digestion by mixing DNA not digested with HpyCH4 and DNA digested with HpyCH4 in a ratio of 100% to 1%. The results are shown in FIG. 5.

The results in FIG. 5 show that amplification of the unaged DNA was completely blocked by the addition of PNA (curve 5), while the cleaved DNA was selectively amplified (curves 1-4). Mixing experiments showed that as low as 1% of the digested DNA in the mixture could be detected. In addition, by calculating the Ct of the amplification of the unaged DNA, as little as 0.01% of the cleaved DNA can be detected.

Example 4

After digesting the DNA with various concentrations of enzyme, PCR was performed, and the results are shown in FIG. 6.

The PCR analysis of the digestion time course is shown in FIG. 7.

The results of FIGS. 6 and 7 show that the concentration of cleaved DNA in the enzymatic reaction gradually increases as the enzyme concentration increases and the reaction time increases until the completion of the enzymatic cleavage. This indicates that enzymatic digestion can be detected quantitatively and that the method can be used to monitor enzymatic digestion up to 100% lysis.