CN1394964A - PCR and short segment DNA sequencing combined diagnosis method - Google Patents

Abstract

The diagnosis method combining PCR and short segment DNA sequencing relates to a biological detection method, specially it is applicable to the research fields of antenated diagnosis of genetic disease, detection of pathogenic pathogen, detection and diagnosis of cancer gene, genetic fingerprinting, individual identification and offspring-parent relationship determination, animal and plant quaretine and biological emdicine, etc. Said invention uses two ends of micro target gene or two-side known nucleotide sequences to design a pair of primers, and uses DNA to be identified as template to make PCR amplification.

Description

Diagnosis method combining polymerase chain reaction with short fragment DNA sequencing

Technical Field

The invention discloses a diagnosis method combining polymerase chain reaction and short fragment DNA sequencing, relates to a biological detection method, and is particularly suitable for the research in the fields of prenatal diagnosis of genetic diseases, detection of pathogenic pathogens, detection and diagnosis of oncogenes, DNA fingerprints, individual identification, paternity testing, forensic physical evidence, animal and plant quarantine, high-tech biomedicine and the like.

Technical Field

In recent years, gene analysis and genetic engineering techniques have revolutionized the development and application of Polymerase Chain Reaction (PCR). The application of PCR technology can make specific gene or DNA fragment in short 2-3 hours in vitro amplification of hundreds of thousands to millions of times. The amplified fragments can be directly visualized by electrophoresis or can be used for further analysis. Thus, a small number of single copies of a gene can be directly observed after amplification to a million fold without the need for isotopic enhancement, and the diagnostic time required to be one or two weeks before can be reduced to hours. The PCR reaction has strong specificity and high sensitivity, and trace DNA can be used as an amplified template to obtain a large number of amplified fragments. The DNA of hair, blood stain, or even single cell can be used for PCR amplification. Therefore, in less than 10 years, the PCR technology has practical value in the fields of prenatal diagnosis of genetic diseases, detection of pathogenic pathogens, detection and diagnosis of oncogenes, DNA fingerprinting, individual identification, paternity testing, forensic physical evidence, animal and plant quarantine, high-tech biomedicine and the like, and the application range of the PCR technology is continuously expanding. However, precisely because of the high specificity and sensitivity of the PCR reaction, unexpected errors often occur in clinical medical diagnosis, leading to unreliable diagnostic results. The most frequently occurring problems are mainly manifested in the following two aspects. First, false negative: the most common causes of false negative results are: the activity of Taq DNA polymerase is insufficient or inhibited; the design of the primers is unreasonable; the quality or quantity of the extracted template is not excessive and the PCR system is not appropriate; the number of cycles is not sufficient. Second, false positive: false positives in PCR results are true for the specimen being tested, and the amplified products in the reaction system. Because PCR technology is highly sensitive, contamination of extremely minute amounts of target gene leads to massive amplification and results misjudgment, and thus contamination is a major source of false positives in PCR. Contamination of PCR is mainly cross-contamination between samples and contamination of amplicons. Another possibility for a false positive result is the presence of homologous sequences of the target gene in the sample. In order to avoid false positives due to contamination, a disposable tip is used to isolate different operating areas, dispense reagents, simplify the operating procedure, and perform PCR.

Of the above two problems, the false negative problem has a lot of experience and solutions in long-term experiments, and generally, the final credible result can be obtained only by examining and verifying the problems in sequence according to various conditions. However, false positive errors occur, and besides contamination is a major factor, defects in the PCR technique itself are also a significant cause. The annealing temperature design is difficult to be precise, and the mispairing and a certain degree of single nucleotide misincorporation of the primer can cause the generation of false positive results. Moreover, finding the cause and correcting it is very time and labor consuming. Therefore, a successful PCR detection method needs to be finally applied to clinic after long-time condition exploration and practical verification, and the requirement on the specification of operation is high in the practical operation process, which limits the application to a certain extent.

Disclosure of Invention

The invention aims to: the method provided retains the characteristics of high sensitivity and strong specificity of the PCR method and can judge whether the target DNA fragment exists in the PCR product.

The purpose of the invention can be realized by the following technical scheme: a method for combining polymerase chain reaction with short segment DNA sequencing designs a pair of primers by using known nucleotide sequences at two ends or two sides of a trace target gene, and uses the DNA to be identified as a template for amplification (PCR method), and is characterized in that: and amplifying again by taking the PCR product as a template, and selecting dozens or even a few of nucleotides which are conservative and have higher specificity in the whole sequence of the corresponding detection object DNA to perform short-fragment DNA sequencing. The phenomena of false negative and false positive which are frequently generated in the prior PCR technology are overcome, and the accuracy is improved. The invention adopts a method of combining a PCR method and short fragment DNA sequencing. On one hand, the characteristics of high sensitivity and strong specificity of the PCR method are retained, on the other hand, by applying the current mature small fragment DNA sequencing method, only dozens or even a few nucleotides which are relatively conservative and have relatively high specificity in the whole sequence of the corresponding detection object DNA are selected, whether the target fragment exists in the PCR product can be judged, and a positive or negative result is determined. The PCR operation is not critical. Because the primer is redesigned in the sequencing process and the PCR product is used as the template for secondary amplification, the false positive rate is greatly reduced, the requirements on the PCR test conditions can be reduced, such as annealing temperature, cycle number and the like are not required to be strict any more, and the groping time and energy are greatly reduced. Due to time and effort. Because the specific region of the related gene is sequenced, the specific sequence of the related gene can be known exactly, the gene can be classified and identified, and the method can be widely applied to various fields.

On the basis of the technical scheme, the primer design in the short-fragment DNA sequencing is designed aiming at the DNA which is related to diseases and has a known gene sequence. Therefore, the method is applied to the multidisciplinary fields of disease diagnosis, species research and the like.

On the basis of the technical scheme, when the sequencing primer is designed according to the known gene sequence related to high-incidence genetic diseases and diseases with genetic tendency, the invention is used for diagnosing the genetic diseases. Generally, a genetic disease can be diagnosed by clinical symptoms, pedigree analysis, and biochemical and immunological detection means, but for diagnosis of heterozygotes in recessive genetic diseases and judgment of whether or not the progeny of heterozygotes are affected (prenatal diagnosis), detection of a disease gene is required. For high-incidence genetic diseases, such as thalassemia, sickle cell anemia, blood coagulation factor deficiency and the like, or genetic-prone diseases, especially part of senile diseases, such as diabetes, hyperlipidemia and eventumors, as long as the gene sequences related to the diseases are known, the method can be used for detecting and rapidly determining the mutation sites or deleted fragments.

On the basis of the technical scheme, a primer or a probe is designed according to the pathogenic gene of exogenous invasion. Once the partial nucleic acid sequence of the exogenous invaded gene is clarified, it can be detected by PCR, RT-PCR or hybridization method, including bacteria, virus, protozoa and parasite, mould, rickettsia, chlamydia and mycoplasma, and the diagnosis by combining PCR with short segment DNA features that the conserved region in the gene can be selected for universal detection and the gene site with great difference can be selected for typing detection. The kit can be used for special detection of a pathogen, and can also be used for multi-element detection of different varieties of related viruses and bacteria. And the accurate DNA sequence can be obtained for analysis, the detection sensitivity and specificity are far higher than those of the current immunological method, and the required time also reaches the clinical requirement, which is particularly suitable for the virus (hepatitis B), bacteria (such as tuberculosis and anaerobe), protozoa (such as treponema pallidum) and the like which are difficult to culture.

On the basis of the technical scheme, by designing specific primers aiming at the oncogene, the anticancer gene and the anti-metastatic gene to carry out PCR amplification and short-segment sequencing, whether the oncogene, the anticancer gene and the anti-metastatic gene are mutated or not can be judged, and the method is used for detecting and diagnosing the oncogene. Although the research on oncogenes is mostly in the fundamental stage, the basic fact that canceration is caused by genetic variation is undoubted, and thus, the research on oncogenes, anticancer genes and anti-metastatic genes cannot be carried out by diagnostic means away from the molecular level, and clinically applicable examples include the quantification of leukemia residual cells (including lentil and acute granulosa), the inactivation of anticancer genes such as P53 and Rb in lung cancer, and the activation and expression of the N-myc gene of a glioma. By designing specific primers for the genes to carry out PCR amplification and short fragment sequencing, whether the oncogene, the anticancer gene and the anti-metastatic gene generate mutation can be judged, so that the gene diagnosis of the tumor is completed, and particularly, the early diagnosis of the tumor is advantageous.

On the basis of the technical scheme, primers are designed aiming at the small myoglobin gene, β -globin gene and ApoB gene, and are applied to identification of DNA fingerprints, individual identification, paternity and species research according to polymorphism and repetition frequency difference.

On the basis of the technical scheme, the primers are designed aiming at the pathogens of the fulminant infectious diseases and are used for whether quarantine personnel, animals and plants carry the pathogens of the fulminant infectious diseases or not and whether food, feed and the like carry salmonella or not. The sensitive, specific and fast diagnosis and detection method for animal and plant quarantine is a necessary guarantee for improving the comprehensive national strength of China by checking whether people, animals, plants (breeding stock, seed) and the like going in and out of China carry virulent infectious disease (AIDS, animal virus, plant virus and the like) pathogens, whether food, feed and the like carry salmonella and the like and needing genetic diagnosis means and rejecting the pathogens out of China.

On the basis of the technical scheme, the invention is used for checking the existence of the implanted gene in the transgenic animal andplant. Plays an important role in the high-tech biomedical field.

The invention has the advantages that: simple operation, time and labor saving, high sensitivity, strong specificity and low requirement on the quality of the original material. The details are given below by way of examples.

The first embodiment is the steps and conditions of the present invention. 1, adding the following components into a typical PCR reaction system: appropriate buffer solution and trace amount of moldPlate DNA, 4 XdNTPs, thermostable polymerase, Mg²⁺And two synthetic DNA primers. Denaturation of the template DNA at 94 ℃ for 1min, annealing of the primer and the template at 40-60 ℃ for 1min, and extension at 72 ℃ for 2 min. Pre-denaturing the template for 3-5 min before the first circulation; after the last cycle, the sample still needs to be extended for more than 3-5 min to ensure that the amplified DNA is double-stranded DNA. 2, selection of sequencing primers: since the complete sequence of DNA is known for clinical genetic screening of diseases, the causative agents of which, such as viral, bacterial or genetic mutations, are known, the tens of base pairs used to determine the presence of the disease should be the most conserved and most specific. SequencingThe amplified fragment should be within this range. The primer selected should also be selected in this region or in the region upstream thereof.

Pyrosequening is described below^TMFor example, the principle and procedure of short-fragment DNA sequencing are described.

The basic principle of Pyrosequencing is to monitor the extension of a nucleic acid strand using a cascade of chemiluminescent reactions. The degree of luminescence detected is directly proportional to the number of incorporated nucleotides. The base sequence ofthe site to be detected can be known by detecting the luminescence of the four nucleotides when added. The method comprises the following specific steps:

the polymerase-catalyzed chain extension reaction of the first DNA step produces a pyrophosphate.

In the second step, pyrophosphoric acid reacts with APS to form ATP under the catalysis of Sulfurylase.

And thirdly, the ATP emits fluorescence under the action of Luciferase (Luciferase).In the fourth step, the intensity of the fluorescence emitted is detected to determine the number of incorporated nucleotides. In the fifth step, unreacted dNTPs are degraded by Apyrse. Second, the use of the invention

The PCR and short-fragment DNA sequencing can be applied to various gene detection fields, and are more accurate and reliable compared with PCR detection, for example, α thalassemia is diagnosed, α barrenness is caused by deletion and point mutation of α globin gene in different ranges, but deletion type mutation is most common, deletion range can be from deletion of two α -genes to deletion of small fragments, and can be detected in people, another mutation of α -gene is single base substitution, more than 18 mutations are found at present, and comprise missense mutation, unintentional mutation splicing site mutation and initial signal mutation, therefore, the main task of α barrenness PCR diagnosis is to quickly detect deletion fragment or mutation site of 23 globin gene, the fact that the deletion or mutation can not be exactly pointed out by using PCR technology, or the fact that a few bases or a certain mutation is deleted is found by only by using a pair of primers, the PCR technology can not accurately judge PC of α barrenness genotype, or the PC can be detected by using a pair of primers, 5, 30 bp more than 30 bp of PCR amplification can be detected by using a pair of primers, and a PCR amplification rate can be greatly reduced by using PCR amplification primer, and a PCR detection result can be found by using a primer pair of PCR detection primer, and a detection result of PCR detection result can be found by using a primer of PCR detection method, wherein the detection result can be found by using a PCR detection method, and a detection result of PCR detection method.

The advantages of the invention are shown in the following aspects:

1, the operation is simple and convenient: the current PCR technology can adopt high temperature resistant Taq DNA polymerase and is carried out in a DNA amplification instrument controlled by a computer, so that the operation is greatly simplified, and the whole reaction process can be satisfied by adding the enzyme once. The DNA amplification instrument can automatically and rapidly raise and lower the temperature, and only all materials required by the reaction need to be uniformly mixed and placed in the instrument, and the reaction is carried out according to the input program. The obtained PCR product can be directly sequenced and analyzed after simple purification, and the sequencing work can be quickly completed by a full-automatic DNA sequencer or a manual sequencing method because only the sequence as short as dozens or even a few of nucleotides needs to be determined.

2, saving time and labor: when Taq DNA polymerase is used, the mononucleotide incorporation rate is high, and at 75-80 ℃, each enzyme molecule can complete the synthesis of 150 nucleotides per second. Each cycle of PCR takes several minutes, so 20-30 cycles are usually used, and the reaction for achieving million times of amplification of the target DNA can be completed in several hours. The target DNA fragment amplified by the PCR method can be directly used for sequence analysis, and a manual sequencing method, namely an isotope labeling method is adopted for short fragment sequence determination; the latest Pyrosequencing can also be applied^TMThe sequencing method monitors the DNA sequence in real time according to the doped dNTP and the fluorescence intensity in the DNA polymerization process, and omits the double eliminationThe PCR amplification step in the oxygen sequencing method can judge the experimental result within dozens of minutes. Also, the most commonly used DNA dideoxy termination method at present is used for sequence analysis because only a few tens of bases of oligonucleotides need to be amplified, a large amount of time can be omitted for the first PCR amplification step, and gel analysis can be performed using a short plate of a few tens of centimetersElectrophoresis, so the experimental results were obtained within a few hours. Because the obtained result has high accuracy, the time for repeated verification is saved, and a large amount of manpower and material resources are saved.

3, high sensitivity: the generation of PCR products is exponentially increased, so that it is not difficult to amplify pg-scale starting materials to μ g levels or to amplify eukaryotic single-copy genes by PCR. PCR methods can be used to perform DNA typing with mono, diploid cells, one hair, and even a single sperm. The fluorescein sequencing method can accurately monitor the nucleotide combined during DNA amplification, and the sensitivity of the method is improved.

4, strong specificity: the correctness of the binding of the oligonucleotide as a primer to the template is critical in determining whether the reaction product is specific. The high temperature resistance of Taq DNA polymerase enables the annealing step of the primer and the template in the reaction to be carried out at higher temperature, the binding specificity is greatly increased, and the amplified target fragment can also keep high accuracy. Meanwhile, the primers selected in the sequencing process are not primers used in PCR, but are redesigned according to highly conserved regions in the corresponding PCR product sequences. Therefore, the method is actually equivalent to performing secondary PCR amplification, and the specificity of the product is greatly improved. But the time is shortened and the cost is reduced compared with the nested PCR or double-PCR. The sequencing result is to verify the specific amplification of the target gene.

5, low requirements on the quality of the raw materials: because of the high sensitivity and specificity of PCR, only a trace amount (pg, ng) of crude product of target DNA or total RNA can be used as the starting material for the reaction to obtain the target product. The partially degraded DNA material can also be subjected to multiple reaction cycles by PCR to finally obtain the desired full-length DNA fragment.

Claims (10)

1. A method for combining polymerase chain reaction with short fragment DNA sequencing designs a pair of primers by using known nucleotide sequences at two ends or two sides of a trace target gene, and uses the DNA to be identified as a template for amplification (PCR method), and is characterized in that: and amplifying again by taking the PCR product as a template, and selecting dozens or even a few of nucleotides which are conservative and have higher specificity in the whole sequence of the corresponding detection object DNA to perform short-fragment DNA sequencing.
2. The method of claim 1, wherein the polymerase chain reaction is combined with short-fragment DNA sequencing, comprising: the design of primers in short-fragment DNA sequencing is designed according to DNA related to diseases and with known gene sequences.
3. A method of combining polymerase chain reaction with short fragment DNA sequencing according to claims 1 and 2, characterized in that: sequencing primers are designed based on known gene sequences associated with high-incidence genetic diseases, genetically predisposed diseases.
4. The method of the combination of polymerase chain reaction and short-fragment DNA sequencing according to claims 1 and 2, characterized in that: and designing a primer or a probe according to the pathogenic gene invaded by the exogenous source.
5. The method of claim 4, wherein the polymerase chain reaction is combined with short-fragment DNA sequencing, and wherein the method comprises the steps of: conserved regions in their genes can be selected for universal detection.
6. The method of claim 4, wherein the polymerase chain reaction is combined with short-fragment DNA sequencing, and wherein the method comprises the steps of: the gene sites with larger differences can be selected for typing detection.
7. Use of the polymerase chain reaction in combination with short-fragment DNA sequencing method according to claims 1 and 2, characterized in that: the mutation of the oncogenes, the anticancer genes and the anti-metastatic genes can be judged by designing specific primers for the oncogenes, the anticancer genes and the anti-metastatic genes to carry out PCR amplification and short fragment sequencing.
8. Use of the polymerase chain reaction in combination with short-fragment DNA sequencing method according to claims 1 and 2, characterized in that primers are designed for myoglobin minor satellite gene, β -globin gene, ApoB gene, and are used for identifying DNA fingerprints, individual identification, paternity and species studies based on the difference in polymorphism and repetition number.
9. Use of the polymerase chain reaction in combination with short-fragment DNA sequencing method according to claims 1 and 2, characterized in that: the primer is designed aiming at the pathogens of the fulminant infectious diseases, and is used for whether quarantine personnel, animals and plants carry the pathogens of the fulminant infectious diseases or not, and whether food, feed and the like carry salmonella or not.
10. Use of the polymerase chain reaction in combination with short-fragment DNA sequencing method according to claims 1 and 2, characterized in that: used for checking the existence of the implanted gene in the transgenic animals and plants.