CN117025734A - Method for improving genotype detection accuracy based on PCR dominant molecular marker - Google Patents

Abstract

The application discloses a method for improving genotype detection accuracy based on PCR dominant molecular markers, which comprises the following steps: 1) When a PCR system is configured, adding an internal reference marker transformed by a housekeeping gene action into the original system to serve as a background band; adding an internal reference mark according to 1/100-1/1 of the content of the dominant primer; 2) Performing PCR operation to obtain an electrophoresis strip result; 3) When the band type is read according to the electrophoresis band result, the band result is analyzed according to the following standard, and the band genotype is judged. The method can solve the problem that the invalid PCR amplification is judged to be the deletion of the ribbon genotype, and improves the detection accuracy of the genotype based on the PCR dominant molecular marker.

Description

Method for improving genotype detection accuracy based on PCR dominant molecular marker

Technical Field

The application relates to the technical field of PCR (polymerase chain reaction), in particular to a method for improving the genotype detection accuracy of a dominant molecular marker based on PCR.

Background

The advent of a range of tools and techniques in the field of molecular biology has provided new opportunities for efficient crop genetic improvement, one of which is the use of molecular markers for genomic mapping (Gene mapping), gene tagging (Gene mapping) and marker assisted selection (Marker assisted selection, MAS).

There may be genetic differences in the DNA nucleotide sequences at corresponding positions on homologous chromosomes between any two different individuals. The molecular marker (Molecular Markers) is a genetic marker based on nucleotide sequence variation or difference in genetic material among individuals, and is a direct reflection of genetic polymorphism at the DNA level.

Depending on the detection method, molecular markers can be broadly divided into three categories: molecular markers based on nucleic acid blot in situ hybridization, molecular markers based on PCR, and molecular markers based on DNA sequencing.

In practical detection, visualization of genetic differential molecular markers can be achieved by gel electrophoresis techniques and chemical-based (ethidium bromide or silver) staining radioactive or colorimetric probe detection techniques. Among them, molecular markers that can reveal differences between individuals of the same or different species are called polymorphic markers.

Depending on whether homozygotes and heterozygotes can be distinguished, polymorphic markers can be distinguished as co-dominant markers or dominant markers as shown in FIG. 1. The co-dominant marker indicates the difference in the size of the band or fragment, while the dominant marker indicates the difference in the presence or absence of the band or fragment.

Many different genotypes may be present on the co-dominant marker (e.g. different size bands on the gel) at the same time, whereas the dominant marker has only two alleles.

The accuracy of genotype detection results of breeding materials relates to the effectiveness of subsequent gene map-based cloning and molecular marker assisted breeding, and false positives or false negatives can lead to the deviation of a positioning interval, can not accurately obtain target site materials without increasing workload, and can influence the normal operation of breeding work.

It is generally recognized that ideal molecular markers should be co-dominant, uniformly distributed throughout the genome, with a high degree of reproducibility and the ability to detect high levels of polymorphisms. The disadvantage of dominant markers over co-dominant markers is not only the inability to distinguish heterozygous genotypes, but also the possibility of determining as missing band genotypes an ineffective PCR amplification caused by any unknown error in the assay procedure.

For example, samples that should have been amplified with bands may exhibit no bands due to insufficient amounts of DNA in a sample, sample omission, sample degradation, enzyme failure, or other unknown reasons. If it is determined that there is no band genotype, a genotype determination error is caused. The above situation is often encountered in practical work and lacks an effective solution.

On the other hand, in some specific cases, only dominant markers are available for selection.

For example, in the DNA molecular marker type, RAPD, AFLP, ISSR and retrotransdonsons converted molecular markers are dominant markers. For co-dominant SSR markers, the markers can be dominant markers under the conditions of detecting penetration of exogenous chromosome fragments of the kindred wild, deletion of large fragments, and the like, and some disease-resistant genes distributed in part of resources only.

Therefore, it is necessary to find a method capable of solving the problem of determining that the invalid PCR amplification is a deletion of the band genotype.

Housekeeping genes refer to a class of genes that are constitutively expressed in cells, the products of which are essential for maintaining various basic vital activities of the cells. The expression level of the housekeeping gene is less affected by environmental factors and can be stably and continuously expressed in all or most tissues of an individual at each growth stage, so that the housekeeping gene is often used as an internal reference gene. For example, "Qin Ziyu. Screening of apple reference genes and establishment of real-time fluorescent quantitative RT-PCR detection methods for three latent viruses TaqMan probes [ D ]. Hebei university of agriculture, 2015". Actin and UBQ were used as preferred reference genes for apples. However, the prior art is mainly used for virus detection, is not used for biological breeding, and cannot effectively solve the problem of false positive in crop breeding.

Disclosure of Invention

The application provides a method for improving the detection accuracy of a genotype based on a PCR dominant molecular marker, which is used for solving the technical problem that invalid PCR amplification is judged to be a missing ribbon genotype in the prior art.

The application provides a method for improving genotype detection accuracy based on PCR dominant molecular markers, which comprises the following steps:

1) When a PCR system is configured, adding an internal reference marker transformed by a housekeeping gene action into the original system to serve as a background band;

adding an internal reference marker transformed by the housekeeping gene action according to 1/100-1/1 of the content of the dominant primer;

adding a housekeeping gene according to 1/10 of the content of the dominant primer;

2) Performing PCR operation to obtain an electrophoresis strip result;

3) When the band type is read according to the electrophoresis band result, the band result is analyzed according to the following standard, and the band genotype is judged to be obtained:

a. the internal reference is marked with a band, the target band is marked with a band, and the band is judged to be of a band gene type;

b. the internal reference mark is provided with a band, the target band is not provided with a band, and the target band is judged to be a non-band gene type;

c. the internal reference mark is free of bands, the target band is provided with bands, and the band-borne base type is judged;

d. the internal reference marker is without a band, and the target band is without a band, and the invalid PCR, namely the deletion, is judged as indicated by "-".

Preferably, the internal reference marker is a housekeeping gene action transformation primer.

Preferably, the housekeeping gene is added at 1/10 of the dominant primer content.

Preferably, the length of the action primer is 250 bp-1000 bp.

Preferably, the reaction system comprises: when the first reaction system and the second reaction system are used, equivalent internal reference markers for the conversion of the housekeeping gene are added into the first reaction system and the second reaction system.

Preferably, the PCR operation comprises the steps of:

(1) carrying out denaturation treatment on the sample at 94 ℃ for 5min;

(2) denaturation at 94℃for 30s;

(3) annealing at 55-60 deg.c for 30s;

(4) extension treatment at 72 ℃ for 30s;

(5) turning to the step (2) and cycling for 35 times, and then entering the next step;

(6) stretching at 72 ℃ for 10min;

(7) hold 12 ℃ and wait for removal.

The application has the beneficial effects that:

1) The method for improving the genotype detection accuracy based on the PCR dominant molecular marker provided by the application can solve the problem that the invalid PCR amplification is judged to be the missing strip genotype, and can improve the genotype detection accuracy based on the PCR dominant molecular marker.

2) According to the method for improving the genotype detection accuracy based on the PCR dominant molecular marker, provided by the application, the obtained invalid PCR amplification can be accurately obtained by adding the housekeeping gene as a reference strip and analyzing the strip condition, so that the erroneous judgment of the invalid PCR is avoided, and the accuracy of judging the strip genotype is improved.

Drawings

FIG. 1 is a schematic diagram showing a comparison of markers for distinguishing homozygotes from heterozygotes co-dominant (a) and dominant (b); co-dominant markers clearly distinguish between homozygotes and heterozygotes, whereas dominant markers are not. The genotypes of the two marker loci are shown below the gel panel (cold et al 2005).

FIG. 2 is a schematic diagram showing a comparison of the method of example 1 of the present application for the resulting co-dominant (a), dominant (b) and modified dominant (c) markers;

FIG. 3 is a comparative chart of the method of the present application applied to polyacrylamide gel electrophoresis in the comparative example, wherein (a) is the result of dominant labeling obtained in the comparative example; (b) a dominant marker obtained by the method of example 1 of the present application;

FIG. 4 is a diagram of dominant marker electrophoresis of the method of the present application applied to agarose gel electrophoresis; red arrows in the figure indicate target strip positions;

FIG. 5 is a diagram of dominant marker electrophoresis using capillary electrophoresis in accordance with the method of the present application; the red arrow in the figure indicates the target strip position.

The results of FIGS. 3, 4 and 5 are combined to demonstrate that the method is applicable to all electrophoresis methods.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. The components of the embodiments of the present application generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the application, as presented in the figures, is not intended to limit the scope of the application, as claimed, but is merely representative of selected embodiments of the application. All other embodiments, based on the embodiments of the application, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the application.

The technical means which are not described in detail and are not used for solving the technical problems of the application are all arranged according to common general knowledge in the field, and various common general knowledge arrangement modes can be realized.

Examples

The instruments used in the following examples and comparative examples are all commercially available products.

Comparative example conventional PCR procedure:

polymerase Chain Reaction (PCR) in Applied9700 PCR apparatus.

The PCR reaction adopts a 10 mu l system, and the specific process is as follows:

1) Composition and configuration system:

depending on the DNA polymerase used, the reaction system 1 component and the reaction system 2 component were used, respectively, and the reaction system components are shown in Table 1. DNA Taq polymerase in Table 1 was purchased from TaKaRa (Dalian); 2X Tag PCR StarMix with Loading Dye, beijing Kang Runcheng (GeneStar) Biotechnology Co., ltd.

The corresponding reaction system of each enzyme is shown in the product instruction book of the purchased enzyme.

2) The reaction procedure: (1) denaturation at 94℃for 5min; (2) denaturation at 94℃for 30s; (3) annealing at 55-60 ℃ for 30s; (4) extending at 72 ℃ for 30s; (5) transferring to the step (2) and cycling for 35 times; (6) extending at 72 ℃ for 10min; (7) hold 12 ℃ and wait for removal.

Selecting the annealing temperature of the step (3) according to the primer specified in each enzyme instruction; markers lacking amplification information may first be initially detected at 55 ℃.

TABLE 1

Example 1 the present application provides a method

The difference from comparative example 1 is that: when a PCR system is configured, an internal reference marker transformed by a house-keeping gene (house-keeping gene) is added as a background band based on the original system, and the house-keeping gene used in the embodiment is an action primer.

An action primer was added at 1/10 of the dominant primer content. The reaction system used is shown in Table 2, and the results obtained are shown in FIG. 2. The indication effect obtained at this time is optimal.

TABLE 2

2. When the tape type is read, judging according to the tape condition:

1) An action is provided with a band, and a target band is provided with a band gene, namely BB;

2) The action has a band, the target band is not provided with a band, and the target band is judged to be provided with a band gene type, namely bb;

3) The action is without band, the target band is judged to be the band gene type, namely BB;

4) An action without band, a target without band, judging as invalid PCR, namely deletion, and representing by "-";

the results obtained in this example are shown in FIG. 2, and the target band is determined to be the band-based type for BB being an action band.

If bb is an action band, the target band is not band, and the target band is determined to be a non-band genotype.

By adopting the method, the condition that the invalid PCR amplification is judged to be the deletion of the ribbon genotype can be effectively avoided.

Example 2 the present application provides a method

The difference from example 1 is that: when a PCR system is configured, the length of the added action primer is 250bp.

Example 3 the present application provides a method

The difference from example 1 is that: when a PCR system is configured, the length of the added action primer is 500bp.

Example 4 the present application provides a method

The difference from example 1 is that: when a PCR system is configured, the length of the added action primer is 1000bp.

Example 5 the present application provides a method

The difference from example 1 is that: the housekeeping gene was added at 1/100 of the dominant primer content.

Example 6 the present application provides a method

The difference from example 1 is that: the housekeeping gene was added at 1/1 of the dominant primer content.

Example 7 the present application provides a method

The difference from example 4 is that: when the PCR system is configured, the length of the added action primer is 1000bp, different electrophoresis systems are selected, and agarose electrophoresis and capillary electrophoresis are respectively selected.

The results obtained in examples 2 to 7 are the same as those in example 1, and are not described here.

Examples were used for the method in example 1 and comparative example 1:

1. polyacrylamide gel electrophoresis experiment

NtCPS2-SNP1F2 is a PCR-based dominant marker transformed by SNP differences for detecting whether a cigar or aromatic smoke material carries an abietyl site. The procedure of example 1 and comparative example 1, respectively, was used for the detection of NtCPS2-SNP1F2, respectively.

The results obtained in comparative example 1 are shown in FIG. 3a, those obtained in example 1 are shown in FIG. 3b, lanes 1 and 2 of FIG. 3b are the bands of the action, the target band; lane 3 is an action without band, target with band; lane 4 is an action band, target band; lanes 13 and 14 are action with band and target band without band. Based on the above results, lane 4 can be judged as invalid PCR.

2. Agarose gel electrophoresis example:

tobacco tomato spotting disease is a serious disease in tobacco production, mr1 and Mr2 are dominant markers closely linked with disease-resistant sites of the tobacco tomato spotting disease respectively, mr1 and Mr2 are respectively positioned on the left wing and the right wing of the disease-resistant sites, and when both markers are detected positive, the detection material can be judged to carry the disease-resistant sites.

Detection of disease-resistant parent and disease-susceptible parent-derived F using two markers ₂ And (3) selecting a reaction system 2 as a PCR reaction system, adding an action primer into the reaction system, and detecting a PCR result by agarose gel electrophoresis.

The results are shown in FIG. 4, in which lanes-1 to-19 are F to be detected ₂ Group material, lane-20, negative control, M100 bp DNA Ladder Marker. Black arrows (marked by an action at the side) indicate an action internal reference band, and the size of the band is 1000bp; the red arrows (with Mr1 and Mr2 marked alongside the arrows respectively) indicate the amplified bands of Mr1 and Mr2, respectively, the amplified band size of Mr1 being about 500bp and the amplified band size of Mr2 being about 750bp.

As can be seen from FIG. 4, the action band indicates whether the PCR reaction is normally performed, and the target band indicates the genotype data of the target site, and the two are combined to jointly judge the genotype results of different samples, so that the genotype misjudgment caused by the PCR reaction error is reduced.

The genotype of each marker is firstly judged, and the genotype data of the Mr1 or Mr2 marker is independently judged by taking an action marker as a reference. And then synthesizing genotype results of the two markers to judge whether the disease-resistant genes are carried or not.

For example, lanes-13 and-15 both show an action site indicating that the PCR reaction is proceeding normally, but since only 13 bands show weak positives at the Mr1 site, 15 bands are negative at the Mr1 site, it is judged that lanes 13/15 do not carry disease resistance sites in the treated sample. As can be seen from FIG. 4, the accuracy of the judgment result can be enhanced by adding an action site.

As compared with the former, it is demonstrated that the method provided by the present application is effective when the method is replaced with an agarose electrophoresis detection method.

3. Capillary electrophoresis example:

the difference from example 1 is that: according to the existing capillary electrophoresis operation, for F ₂ The group sample is treated, the molecular weight of the sample is 50-100 ng/. Mu.L, the reaction system is reaction system 2, and an action primer is added into the reaction system.

The results are shown in FIG. 5, and it is understood that the method is also applicable to a method for obtaining genotype data by capillary electrophoresis.

This example, in combination with the former two, collectively demonstrates that the method is applicable to each electrophoresis method.

Although the present application has been described with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements and changes may be made without departing from the spirit and principles of the present application.

Claims (6)

1. The method for improving the genotype detection accuracy based on the PCR dominant molecular marker is characterized by comprising the following steps of:

1) When a PCR system is configured, adding an internal reference marker transformed by a housekeeping gene action into the original system to serve as a background band;

adding an internal reference marker transformed by the housekeeping gene action according to 1/100-1/1 of the content of the dominant primer;

adding a housekeeping gene according to 1/10 of the content of the dominant primer;

2) Performing PCR operation to obtain an electrophoresis strip result;

3) When the band type is read according to the electrophoresis band result, the band result is analyzed according to the following standard, and the band genotype is judged to be obtained:

a. the internal reference is marked with a band, the target band is marked with a band, and the band is judged to be of a band gene type;

b. the internal reference mark is provided with a band, the target band is not provided with a band, and the target band is judged to be a non-band gene type;

c. the internal reference mark is free of bands, the target band is provided with bands, and the band-borne base type is judged;

d. the internal reference marker is without a band, and the target band is without a band, and the invalid PCR, namely the deletion, is judged as indicated by "-".

2. The method for improving the detection accuracy of a genotype of a dominant molecular marker based on PCR of claim 1, wherein the internal reference marker is a housekeeping gene action conversion primer.

3. The method for improving the detection accuracy of genotype based on PCR dominant molecular marker of claim 1, wherein the housekeeping gene is added according to 1/100-1/1 of the dominant primer content.

4. The method for improving the detection accuracy of the genotype of the dominant molecular marker based on PCR according to claim 1, wherein the length of the action primer is 250 bp-1000 bp.

5. The method for improving the detection accuracy of a genotype based on a PCR dominant molecular marker of claim 1, wherein the reaction system comprises: when the first reaction system and the second reaction system are used, equivalent internal reference markers for the conversion of the housekeeping gene are added into the first reaction system and the second reaction system.

6. The method for improving the accuracy of genotype detection based on PCR dominant molecular markers of claim 1, wherein the PCR operation comprises the steps of:

(1) carrying out denaturation treatment on the sample at 94 ℃ for 5min;

(2) denaturation at 94℃for 30s;

(3) annealing at 55-60 deg.c for 30s;

(4) extension treatment at 72 ℃ for 30s;

(5) turning to the step (2) and cycling for 35 times, and then entering the next step;

(6) stretching at 72 ℃ for 10min;

(7) hold 12 ℃ and wait for removal.