CN101250586B - Method for detecting trace DNA - Google Patents

Abstract

The present invention relates to a method for micro-DNA detection, a method for reusing the original DNA template after PCR amplification by using ethanol precipitation to overcome the lack of template, which can be used for forensic medicine, paleontology, prenatal diagnosis genetics detection These fields, such as the study of disease genomics, have relatively few DNA samples. Compared with previous methods for detecting trace amounts of DNA, the present invention solves the detection of trace amounts of DNA from the perspective of starting templates, and the entire detection scheme is simple to operate, does not require professionals, has high specificity, and has high sensitivity and PCR efficiency , has broad application prospects in practical applications.

Description

A method for micro-DNA detection

technical field

The invention belongs to the field of nucleic acid detection, in particular to a nucleic acid detection method for recovering and reusing the original DNA template after PCR amplification by ethanol precipitation.

Background technique

In forensic science, paleontology, prenatal diagnostic genetic testing and disease genomics research, it is often impossible to perform effective testing due to the extremely limited DNA content of the sample. Therefore, the detection of trace amounts of template DNA is a difficult problem that needs to be solved urgently in this field.

In order to solve this problem, many researchers try to adopt various methods to improve the detection means to increase the sensitivity of detection. Judging from the current research, almost all related research adopts a series of methods based on polymerase chain reaction (polymerase chain reaction, PCR). To sum up the current research situation, there are two basic strategies, that is, to increase the amount of information that can be obtained by one test, or to conduct research on genetic information after generally amplifying the DNA region to be studied.

For the strategy of increasing the amount of information that can be obtained by one test, the more successful and representative one is multiplex PCR (multiplex PCR). Through a series of optimized combinations, it can analyze multiple genetic information in one PCR reaction and subsequent analysis, which is a high-throughput analysis technology. However, the multiplex PCR amplification technology has problems such as system optimization difficulties, mutual influence of multiple pairs of primers during extension, and signal interference during product analysis. It is also necessary to consider avoiding fragment crossover during detection, chromosome positioning, target gene size, and gene linkage. (J Soc Biol, 2003, 197:351-359). Therefore, in a multiplex PCR amplification system, the number of loci that can actually be detected at one time is limited, and it is often difficult to achieve the purpose of micro-DNA detection. Even if the detection is successful, the sample is often exhausted, and the conditions for re-examination are lost, making the results more reliable. reduce.

For the strategy of studying genetic information after general amplification of the DNA region to be studied, nested PCR technology and whole genome amplification technology are more representative at present. The basic principle of nested PCR (nested PCR) technology is to amplify a longer DNA fragment first, and then perform secondary amplification analysis on the shorter DNA sequence located in this fragment, thereby greatly improving the detection of some The sensitivity and specificity of the ability to detect DNA fragments, but this method is limited to single-site analysis and cannot meet the needs of multi-site multiplex amplification (J Forensic Sci, 1998, 43: 696-700). Whole genome amplification (WGA) is a technical method gradually developed in recent years, which can effectively solve the shortcomings of multiplex PCR and nested PCR. WGA is a group of non-selective amplification techniques for the entire gene sequence, and its purpose is to greatly increase the total amount of DNA without sequence preference. The basic idea is to amplify the entire genomic DNA of a trace tissue or even a single cell, then divide the amplified product into multiple parts, and use it as a template for subsequent analysis, and then complete the analysis of multi-site, multi-gene, and whole-genome DNA. Multiple studies are required (Genome Research, 2003, 13:954-946). Although this method has been widely used in forensic science, the diagnosis of single-gene genetic diseases, and the analysis of disease genes, and has achieved good results, the following problems still need to be solved according to the current reports: WGA technology cannot To achieve 100% amplification of the whole genome, it can only be as close as possible, so we must devote ourselves to finding methods and technologies to achieve the largest range of amplification; any amplification has a certain degree of selective amplification and wrong amplification products. When only a very small amount or even a few cells are used as templates, the occurrence of this phenomenon may cause fatal errors; WGA is only a general term for a type of technology, which includes many specific technologies. WGA techniques may be different, so in practical applications, which specific WGA technique to use and which specific sample problem to solve requires exploratory research.

Contents of the invention

The purpose of the present invention is to provide a method for micro-DNA detection, which can be reused by recovering the original DNA template, which not only overcomes the problems of forensic identification, paleontology, genetic detection of prenatal diagnosis, and research of disease genomics. Due to the problem of insufficient identified templates, it also solved the fidelity problem in WGA from the source. The entire detection scheme is simple to operate, does not require professionals, has high specificity, high sensitivity and PCR efficiency, and has broad application prospects in practical applications.

A detection method of a trace amount of DNA of the present invention is to recover the original DNA template after PCR amplification by ethanol precipitation, and then use the recovered template to continue subsequent detection, which greatly improves the detection efficiency. The specific steps include:

(1) Extraction of DNA from the sample to be tested;

(2) using the DNA in step (1) as a template to carry out the first PCR reaction;

(3) After the reaction is over, take out a part of the PCR reaction solution in step (2) and leave it for the following detection experiment;

(4) Utilizing ethanol precipitation and purification to recover the remaining part of the product in step (2), the original DNA template used for detection;

(5) Carry out the second PCR reaction with the original DNA recovered in step (4) as the initial template;

(6) Repeat (3), (4), (5) as needed;

(7) Perform nested PCR (nested PCR) on the first and second PCR reaction products, and then analyze the results.

The sample DNA can be extracted by using a conventional phenol-chloroform extraction method, or by using a kit.

The first PCR reaction and the second PCR reaction are single or multiplex PCR, and the difference between the first PCR reaction and the second PCR reaction is that the amplified target fragments are different, and the second PCR reaction is different to the first PCR reaction. Genes that were not amplified in the second PCR reaction were amplified.

In the step (4), ethanol precipitation, purification and recovery are carried out by adding absolute ethanol to precipitate at -20 ° C, centrifuging at 12000 rpm for 5 minutes, removing the supernatant, and then washing the precipitated template DNA with 100 μl of 75% absolute ethanol .

Beneficial effects of the present invention:

(1) Repeated use of trace templates to overcome the problem of insufficient templates;

(2) High specificity and high sensitivity;

(3) Low cost;

(4) The operation process of the whole detection scheme is simple and does not require professionals.

Description of drawings

Fig. 1 is the schematic diagram of recovering the original DNA template after PCR amplification by ethanol precipitation, according to the mark shown in the figure, (A) is the remaining product after taking out a small part of the product of the first PCR reaction; (B) is the previous product Add 100 μl of absolute ethanol to (A), precipitate at -20°C, centrifuge at 12,000 rpm for 5 minutes, and remove the supernatant; (C) wash the precipitated template in (B) with 100 μl of 75% absolute ethanol DNA was centrifuged at 12000 rpm for 5 minutes, and the supernatant was removed; (D) was to add a new reaction system to (C) for the second reaction.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

Breast cancer needle biopsy tissue detection.

(1) Genomic DNA was extracted from breast cancer biopsies by conventional phenol-chloroform extraction.

(2) Design three pairs of primers to amplify three fragments on the BRCA1 gene. The primer sequences are as follows: 5'AATGGAAAGCTTCTCAAAGTA 3' on P1, 5'ATGTTGGAGCTAGGTCCTTAC 3' on P1; 5'CTAACCTGAATTATCACTATC 3' on P2, 5'GTGTATAAATGCCTGTATGCA on P2 3'; 5' AATTCTTAACAGAGACCAGAAC 3' on P3, 5' AAAACTCTTTTCCAGAATGTTGT 3' on P3, and each of these primers was diluted to 10 p/mol.

(3) Take the DNA extracted from the tissue, primers P1 up and P1 down, high temperature polymerase 0.5U, and high temperature polymerase buffer for mixed reaction. The reaction conditions are: 95°C for 15 minutes (94°C for 30 seconds, 55°C for 45 seconds, 72°C °C for 45 seconds) 35 cycles at 72 °C for 10 minutes.

(4) Take out a small amount of PCR reaction solution in step (3) for the subsequent nested PCR, and use ethanol precipitation to recover the DNA extracted from the tissue: ethanol precipitation, centrifuge at 12000 rpm for 5 minutes, wash with 75% ethanol, and obtain the initial template.

(5) Take primers P2 up and P2 down, high-temperature polymerase 0.5U, ethanol precipitation to re-recover the template (obtained in step 4), and high-temperature polymerase buffer for mixed reaction. The reaction conditions are: 95°C for 15 minutes (94°C for 30 seconds, 55°C for 45 seconds, 72°C for 45 seconds) 35 cycles of 72°C for 10 minutes.

(6) Take out a small amount of PCR reaction solution in step (5) for the subsequent nested PCR, and use ethanol precipitation to recover the DNA extracted from the tissue: ethanol precipitation, centrifuge at 12000 rpm for 5 minutes, wash with 75% ethanol, and obtain the initial template.

(7) Take primers P3 up and P3 down, high-temperature polymerase 0.5U, ethanol precipitation and re-recovery template (obtained in step 6), and high-temperature polymerase buffer for mixed reaction. The reaction conditions are: 95°C for 15 minutes (94°C for 30 seconds, 55°C for 45 seconds, 72°C for 45 seconds) 35 cycles of 72°C for 10 minutes.

(8) Dilute the fragments P1, P2, and P3 amplified by PCR 1000 times, and use this as a template to perform nested PCR with primers for detection.

Example 2

Mitochondrial polymorphism detection in exfoliated cells of oral mucosa.

(1) Collect oral cotton swabs from 50 volunteers, and use a kit to extract DNA.

(2) Design six pairs of primers to amplify six fragments on the mitochondria. The primer sequences are as follows: 5'CTCCACCATTAGCACCCAAAGC3' on P1, 5'CCTGAAGTAGGAACCAGATG3' on P1; 5'GGTCTATCACCCCTATTAACCAC3' on P2, 5'GTTAAAAGTGCATACCGCCA3' on P2; 5'TGGCCCAACCCGTCATCTAC3', 5'GGAATGCGGTAGTAGTTAGG3' under P3; 5'TGACAAAAACTAGCCCCCAT3' above P4, 5'GCGATGAGTGTGGGGAGGAA3' below P4; 'TTCGCAGGCGGCAAAGACTA3'; and each of these primers was diluted to 10 p/mol.

(3) Take DNA extracted from oral cotton swab, primers P1 upper, P1 lower, P2 upper, P2 lower, P3 upper, P3 lower, high temperature polymerase 0.5U, high temperature polymerase buffer and mix reaction, the reaction conditions are: 95°C for 15 minutes (94°C for 40 seconds, 60°C for 60 seconds, 72°C for 60 seconds) 35 cycles of 72°C for 10 minutes.

(4) Take out a small amount of the product of the first PCR reaction for subsequent detection, and use ethanol precipitation for the remaining part of the product to extract the DNA from the oral cotton swab: ethanol precipitation, centrifuge at 12000 rpm for 5 minutes, wash with 75% ethanol to obtain the initial template .

(5) Take primers P4 up, P4 down, P5 up, P5 down, P6 up, P6 down, high temperature polymerase 0.5U, ethanol precipitation and heavy recovery template (obtained in step 4), high temperature polymerase buffer mixed reaction, reaction conditions It is: 95°C for 15 minutes (94°C for 30 seconds, 55°C for 45 seconds, 72°C for 45 seconds) and 35 cycles of 72°C for 10 minutes.

(6) Dilute the PCR product of the first reaction and the PCR product of the second reaction by 1000 times, use this as a template to perform nested PCR with primers, and perform sequencing detection on the results of the nested PCR. According to the information of the sequencing results, Mitochondria were polymorphically typed.

Claims (4)

1. A method for trace DNA detection, the steps comprising: (1) Extraction of DNA from the sample to be tested; (2) using the DNA in step (1) as a template to carry out the first PCR reaction; (3) After the reaction is over, take out a part of the PCR reaction solution in step (2) and leave it for the subsequent nested PCR reaction; (4) Utilizing ethanol precipitation and purification to recover the remaining part of the product in step (2), the original DNA template used for detection; (5) Carry out the second PCR reaction with the original DNA recovered in step (4) as the initial template; (6) Repeat (3), (4), (5) as needed; (7) Perform nested PCR on the first and second PCR reaction products, and then analyze the results. 2. The method for micro-DNA detection according to claim 1, characterized in that: said sample DNA is extracted by conventional phenol-chloroform extraction or extraction with a kit. 3. the method for trace DNA detection according to claim 1, is characterized in that: described PCR reaction for the first time and PCR reaction for the second time are single multiplex or multiplex PCR, its PCR reaction for the first time and PCR reaction for the second time The difference in the reactions lies in the fact that the amplified target fragments are different, and the second PCR reaction amplifies the genes not amplified in the first PCR reaction. 4. The method for trace DNA detection according to claim 1, characterized in that: in the step (4), ethanol precipitation, purification recovery is by adding absolute ethanol-20 ℃ precipitation, 12000 rpm/centrifugation for 5 minutes, Remove the supernatant, and then wash the precipitated template DNA with 100 μl 75% absolute ethanol.

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