CN109337973B - Primer design method, primer, kit, method and system for verifying copy number variation of specific DNA fragment - Google Patents

Abstract

The present invention provides a primer design method for verifying the copy number variation of a specific DNA segment, and also provides a primer, a method and a system for verifying the copy number variation of a specific DNA segment. Compared with the prior art, the present invention provides In the primer design method of the invention, the area for designing primers is increased, and is no longer limited to 80bp to 150bp, and the difficulty of primer design is greatly reduced. The sequencing result in the verification method of the present invention is very intuitive, and it is convenient to judge the deletion variation. The primer design method, verification primer, verification method and verification system of the present invention can avoid the influence of homology on verification; the verification method and verification system of the present invention can not only determine whether CNV exists, but also determine whether CNV and minor variation exist. on the same chromosome.

Description

A primer design method, primer, kit, method and system for verifying copy number variation of a specific DNA fragment

technical field

The present invention relates to a primer design method, primer, kit, method and system for verifying the copy number variation of a specific DNA fragment.

Background technique

There are 46 human chromosomes, of which 22 pairs of autosomes appear in pairs. In each pair of chromosomes, one comes from the father and the other is from the mother, called homologous chromosomes. Chromosomes are mainly composed of DNA and proteins, of which DNA or deoxyribose nucleic acid sequence (also known as nucleotide sequence or base sequence) determines the genetic information. DNA is a long-chain polymer composed of four deoxynucleotides, namely adenine deoxynucleotide (dAMP), thymidine deoxynucleotide (dTMP), cytosine deoxynucleotide (dCMP), Guanine deoxynucleotides (dGMP). What determines the diversity of organisms is the four bases in deoxynucleotides, adenine (A), thymine (thymine, T), cytosine (cytosine, C) and guanine (guanine, G), DNA The sequence is usually represented by the sequence of four bases of ATCG, which is called the base sequence or nucleotide sequence, A pairs with T, C pairs with G, and the forward direction refers to the 5' end to 3' end direction.

Gene variation refers to the heritable variation in the genomic DNA molecule. According to the size of the fragment involved in the variation, it can be divided into small variation and copy number variation. Copy number variations (CNVs) refer to DNA fragments longer than 1 kb. Deletion or duplication variant. There are various methods for CNVs detection, such as chromosomal microarray (CMA), single nucleotide genotyping, Multiplex ligation-dependent probe amplification (MLPA) and currently High-throughput sequencing technology widely used in clinical research.

For CNVs detected by CMA and high-throughput sequencing, other methods often need to be further verified. Commonly used methods include MLPA and semi-quantitative qPCR. Among them, qPCR is one of the most commonly used methods. However, the length of qPCR amplification is strict, usually 80bp to 150bp. When the target fragment has high homology, primer design is difficult and it is very easy to fail, and then the verification fails. MLPA is a finished reagent developed by a reagent company, and most genes do not have corresponding probes. In one case, a heterozygous minor variant is detected, and at the same time, a heterozygous deletion CNV is detected nearby. At this time, when the homology is high, it is often difficult to verify the existence of the CNV by qPCR.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a primer design method for verifying the copy number variation of a specific DNA fragment. Based on the primer design method, the present invention also provides a primer, a kit and a method for verifying the copy number variation of a specific DNA fragment. and system to solve the problems raised in the above background art.

In order to achieve the above purpose, the adopted technical scheme: a primer design method for verifying the copy number variation of a specific DNA fragment, the primer design method comprising the following steps:

Design at least a pair of primers for the detection of the heterozygous minor variation for a specific DNA fragment to be verified that is known to have a heterozygous minor variation and may have a heterozygous deletion copy number variation, wherein one primer in the designed primer pair is located in Upstream or downstream of the minor variation, another primer is located in the region to be verified for missing copy number variation, and the designed primer pair is the primer used to verify the copy number variation of the DNA fragment.

Heterozygous minor variation refers to the presence of a minor variation on one chromosome in the homologous chromosome, but the minor variation does not exist at the corresponding position on the other chromosome. Heterozygous deletion copy number variation refers to the presence of deletion copy number variation on one chromosome in homologous chromosomes, but the absence of the deletion copy number variation at the corresponding position of the other chromosome.

The primer of the designed DNA is a small piece of single-stranded DNA, the upstream primer (ie, the forward primer) is complementary to the DNA positive strand (ie, the sense strand), and the downstream primer (ie, the reverse primer) is complementary to the DNA positive strand (ie, there is a positive strand). The primer design generally requires that the length of the primer is 15 to 30 bases, the primer cannot have primer-dimer and hairpin structure, and the primer must have specificity, that is, the conserved region of the DNA sequence. In the present invention, the method for designing primers for a specific DNA fragment to be verified is that one primer in the designed primer pair is located upstream or downstream of the minor variation, and the other primer is located in the CNV region to be verified. Specifically, when the CNV region is located upstream of the minor variation, one primer in the designed primer pair is located downstream of the minor variation, and the other primer is located in the CNV region to be verified; when the CNV region is located downstream of the minor variation, the designed primer pair is in the One of the primers is located upstream of the minor variation, and the other primer is located in the CNV region to be verified; one of the advantages of this design is that it can amplify the minor variation and achieve the purpose of detecting the minor variation.

Preferably, the distance between the minor variation and the deletion copy number variation region in the specific DNA fragment to be verified is less than 800 bp.

The specific DNA fragment to be verified in the present invention is a DNA fragment with heterozygous minor variation and possible heterozygous deletion copy number variation detected by the prior art. Generally, the specific DNA fragment to be verified is a DNA fragment that has been detected by high-throughput sequencing to have a heterozygous minor variation and possibly a heterozygous deletion copy number variation.

Preferably, the designed primer pairs are two pairs, which are respectively an outer primer pair and an inner primer pair for nested PCR amplification. The fusion nested PCR technology in the primer design method of the present invention can avoid the influence of homology on verification.

Preferably, the specific DNA fragment to be verified is the CYP11B1 gene, the heterozygous minor variation is located in the CYP11B1 gene exon 8 c.1391_1393dup, and there may be a heterozygous deletion copy number variation near the upstream of the heterozygous minor variation.

The present invention provides a primer for verifying the copy number variation of a specific DNA fragment, the primer is designed by using the above-mentioned primer design method.

Preferably, the primers consist of an outer primer pair and an inner primer pair amplified by nested PCR:

Outer primer pair:

Upstream primer F1: 5'-TACTTGGGATTGTGATGTG-3' (SEQ ID NO: 1),

Downstream primer R1: 5'-AAACCACAGCACCCTTGCATGGCCA-3' (SEQ ID NO:2);

Inside primer pair:

Upstream primer F2: 5'-TAAACAGCGTCACCCAGCAG-3' (SEQ ID NO:3),

Downstream primer R2: 5'-CTTAGCCTGGCAAACCCTGG-3' (SEQ ID NO:4).

The present invention provides a kit for verifying copy number variation of a specific DNA segment, the kit includes the primers described above.

The present invention provides a method for verifying copy number variation of a specific DNA segment, the method comprising the steps of:

(1) Using a DNA sample containing a specific DNA fragment to be verified that is known to have heterozygous minor variation and may have heterozygous deletion copy number variation as a template, the primers designed by the above-mentioned primer design method are used to amplify, and the The amplified product was subjected to sanger sequencing;

(2) compare the sanger sequencing result obtained in step (1) with the normal human control sequence; after the comparison, the verification results are as follows:

If the sanger sequencing result shows that the minor variation in the specific DNA fragment to be verified is a homozygous minor variation, the specific DNA fragment to be verified has a heterozygous deletion copy number variation and is located on a different homologous chromosome from the minor variation;

If the sanger sequencing result shows that the minor variation in the specific DNA fragment to be verified is a heterozygous minor variation, then the specific DNA fragment to be verified does not have deletion copy number variation;

If the result of sanger sequencing shows that no minor variation is detected in the specific DNA fragment to be verified, the specific DNA fragment to be verified has a heterozygous deletion copy number variation and is located on the same homologous chromosome as the minor variation.

The Sanger method is based on nucleotides starting at a fixed point, randomly ending at a specific base, and fluorescently labeling each base to generate four groups ending with A, T, C, and G. A method of obtaining visible DNA base sequences by electrophoresis on a urea-denaturing PAGE gel for a series of nucleotides of different lengths.

Preferably, the homology between the specific DNA fragment to be verified and other DNA sequences in the DNA sample is greater than or equal to 80%. More preferably, the homology between the specific DNA fragment to be verified and other DNA sequences in the DNA sample is greater than or equal to 90%. When the homology between the specific DNA fragment to be verified and other DNA sequences in the DNA sample is greater than or equal to 80%, it is difficult to design suitable primers using qPCR, especially when the specific DNA fragment to be verified is identical to the When the homology of other DNA sequences in the DNA sample is greater than or equal to 90%, it is more difficult to design suitable qPCR primers.

The present invention provides a system for verifying copy number variation of a specific DNA segment, the system comprising

A gene amplification device for amplifying a DNA sample including a specific DNA fragment to be verified that is known to have heterozygous minor variation and may have heterozygous deletion copy number variation, and the primers used in the amplification For the primers designed by the above-mentioned primer design method;

a gene sequencing device, the gene sequencing device is connected to the gene amplification device, and is used to perform sanger sequencing on the amplified product, so as to obtain a sanger sequencing sequence of minor variation in the specific DNA fragment to be verified;

An alignment device, the alignment device is connected to the genome sequencing device, the normal human reference sequence information is pre-stored in the alignment device, and the alignment device is used to compare the obtained sanger sequencing sequence with the normal human reference sequence Sequence information is compared, and based on the comparison results, the minor variations determined by the sanger sequencing results are obtained, and then combined with the known information of the existence of heterozygous minor variations in the specific DNA fragment to be verified, the heterozygosity in the specific DNA fragment to be verified is verified. The case of deletion of copy number variation; if the sanger sequencing result shows that the minor variation in the specific DNA fragment to be verified is a homozygous minor variation, the specific DNA fragment to be verified has a heterozygous deletion copy number variation and is located at a different location from the minor variation on the homologous chromosome; if the sanger sequencing result shows that the minor variation in the specific DNA fragment to be verified is a heterozygous minor variation, the specific DNA fragment to be verified does not have deletion copy number variation; If no minor variation is detected in the specific DNA fragment to be verified, the specific DNA fragment to be verified has a heterozygous deletion copy number variation and is located on the same homologous chromosome as the minor variation; and

A result output device, which is connected to the comparison device, so as to output a verification result.

Beneficial effects: the present invention provides a primer design method for verifying the copy number variation of a specific DNA fragment, and the present invention also provides a primer, method and system for verifying the copy number variation of a specific DNA fragment, compared with In the prior art, in the primer design method of the present invention, the area for designing primers is increased, and is no longer limited to 80bp to 150bp, and the difficulty of primer design is greatly reduced. The sequencing result in the verification method of the present invention is very intuitive, and it is convenient to judge the deletion variation. The primer design method, verification primer, verification method and verification system of the present invention can avoid the influence of homology on verification; the verification method and verification system of the present invention can not only determine whether CNV exists, but also determine whether CNV and minor variation exist. on the same chromosome.

Description of drawings

1 is a schematic diagram of the design of primers for specific DNA fragment copy number variation in Example 1 of the present invention, in the figure: 1, upstream primer F2; 2, homologous chromosome A; 3, homologous chromosome B; 4, the deletion to be verified Region; 5. Heterozygous minor variation; 6. Downstream primer R2;

Figure 2 is a map of the sequencing results of the upstream primer F2 in Example 1 of the present invention, in the figure: 1, normal human reference sequence; 2, sample sequence; 3, minor variation c.1391_1393dup;

Figure 3 is a map of the sequencing results of the middle and downstream primer R2 in Example 1 of the present invention, in the figure: 1, the normal human reference sequence; 2, the sample sequence; 3, the minor variation c.1391_1393dup.

Detailed ways

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific embodiments.

Example 1

We detected a heterozygous minor variant CYP11B1 gene (reference transcript NM_000497.3) exon c.1391_1393dup by high-throughput sequencing, located on homologous chromosome A, and a heterozygous deletion of a nucleotide sequence near the upstream , the deletion range is about 37kb, and the position is chr8:143956640-143994311. This fragment contains the exons 1-7 of the CYP11B1 gene. Next, the heterozygous deletion fragment is verified. I tried to design primers by qPCR and found that due to the existence of the pseudogene CYP11B2 in the CYP11B1 gene, the homology is very high, reaching more than 90%. However, qPCR has high requirements, and the amplification length is generally required to be 80bp to 150bp. It is difficult to design suitable primers. We Attempts have been made to design several pairs of qPCR primers in the deletion region to be detected; for example: one of the primers: upstream primer 5'-TGGCAACAGGAGAGACAGGAT-3' (SEQ ID NO: 5), downstream primer: 5'-GGCTCCGTATCAACCAGAGAAA-3' (SEQ ID NO:6), the second primer: upstream primer 5'-TCCACCGTCCAGCTCATGT-3' (SEQ ID NO:7), downstream primer: 5'-GCCTCAAAGTGCTCCTTCCA-3' (SEQ ID NO:8), the third primer: upstream primer 5 '-AGGACGTGGAGAAGCTGCAA-3' (SEQ ID NO: 9), downstream primer: 5'-TGCCCACGATGTTGTCTGTAG-3' (SEQ ID NO: 10). Because these primers and amplification regions are located in regions with homologous sequences, the quantitative amplification of the region to be tested will affect the quantitative amplification of the region to be tested, and the state of deletion cannot be correctly determined, and the qPCR experiment fails.

Next, we take the method of the present invention to verify, and the process is described as follows:

First, primers were designed downstream of the deletion region and the small variant c.1391_1393dup detected by high-throughput sequencing:

Upstream primer F1: 5'-TACTTGGGATTGTGATGTG-3' (SEQ ID NO: 1)

Downstream primer R1: 5'-AAACCACAGCACCCTTGCATGGCCA-3' (SEQ ID NO:2)

Using this pair of primers, using the original high-throughput sequencing-detected sample DNA as a template, long PCR was performed to obtain the product P. Among them, the reaction system settings are shown in Table 1:

Table 1 The first round PCR reaction system

reagent Volume (μl) ddH₂O 12.5 2X Phusion Green HS II HF Master Mix 25 Forward primer F1 2.5 reverse primer R1 2.5 DNA template 4 DMSO (3%) 1.5

The PCR reaction program settings are shown in Table 2:

Table 2 The first round PCR reaction program

By performing long PCR, the interference of highly homologous fragments such as pseudogenes can be avoided.

Then, primers were designed in the deletion region detected by high-throughput sequencing and downstream of the minor variation c.1391_1393dup and in the amplification region of primer F1 and primer R1, as shown in Figure 1:

Upstream primer F2: 5'-TAAACAGCGTCACCCAGCAG-3' (SEQ ID NO:3)

Downstream primer R2: 5'-CTTAGCCTGGCAAACCCTGG-3' (SEQ ID NO:4)

The downstream primer R2 is located downstream of the minor variation, and the upstream primer F2 is located in the CNV region to be verified.

Using this pair of primers, PCR amplification was carried out with the long PCR product P as a template, and the reaction system was set as shown in Table 3:

Table 3 Second round PCR reaction system

The reaction conditions are shown in Table 4:

Table 4 The second round PCR reaction program

temperature time Number of reaction cycles 98℃ 3min 1 98℃ 10s 30 63℃ 30s 1 72℃ 1min 1 72℃ 5min 1 25℃ ∞ 1

Subsequently, the PCR product was subjected to sanger sequencing, and the sequencing primers were the upstream primer F2 and the downstream primer R2, and the results shown in Figure 2 and Figure 3 were obtained: the sequencing results of the upstream primer F2 and the downstream primer R2, and the comparison between the sample sequence and the normal human reference sequence, both The minor variant c.1391_1393dup was shown to be homozygous.

Finally, the results are analyzed as follows:

(1) If the detected minor variant c.1391_1393dup is homozygous: it is assumed that CNV (chr8:143956640-143994311) exists and is located on a different homologous chromosome from the minor variant.

(2) If the detected minor variation c.1391_1393dup is heterozygous: it is presumed that the CNV does not exist.

(3) The minor variation c.1391_1393dup was not detected, that is, only the wild type was detected: it is presumed that the CNV exists and is located on the same homologous chromosome as the minor variation.

The results detected in this experiment belong to the first case, that is, the minor variant c.1391_1393dup is homozygous. Combined with the high-throughput sequencing of the minor variant c.1391_1393dup heterozygous, it is speculated that another homologous chromosome segment of the minor variant is homologous chromosome B. Because the deletion was not amplified, there was no sequencing data. Therefore, this sanger sequencing result supports the deletion variant detected by high-throughput sequencing. Verification succeeded.

In the embodiment of the present invention, the long PCR amplification of the primers F1 and R1 can avoid the influence of homologous sequences, and the homology is not high, such as less than 80%, long PCR can be omitted, and this step is ignored. Verification of CNV by sanger sequencing method effectively reduces the limitations of PCR primer design. At the same time, it can be speculated whether the adjacent small variation and CNV are on the same chromosome, which may have certain significance for the interpretation of autosomal recessive genetic diseases.

In contrast, qPCR has high design requirements, short amplification length, strict requirements, high difficulty in primer design, and relatively poor experimental stability. When the homology is high, it is often difficult to design suitable primers, so it is difficult to carry out verification experiments. Since qPCR is a semi-quantitative detection of the amplification of the region to be tested, it is impossible to know the relative position of the deletion region and the minor variation, that is, whether they are on the same homologous chromosome.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that, The technical solutions of the present invention may be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

sequence listing

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Claims (7)

1. A primer design method for verifying copy number variation of a specific DNA fragment, comprising the steps of:

aiming at a specific DNA fragment to be verified, wherein the specific DNA fragment is known to have heterozygous minor variation and possible heterozygous deletion copy number variation, designing at least one pair of primers for detecting the heterozygous minor variation, wherein one primer in the designed primer pair is positioned at the upstream or downstream of the minor variation, the other primer is positioned in a deletion copy number variation area to be verified, and the designed primer pair is used for verifying the copy number variation of the specific DNA fragment;

the specific DNA fragment to be verified is CYP11B1 gene, the heterozygous minor variation is positioned at exon 8 c.1391 — 1393dup of CYP11B1 gene, and the heterozygous deletion copy number variation possibly exists near the upstream of the heterozygous minor variation;

the primers used to verify the copy number variation of a specific DNA fragment consist of the outside primer pair and the inside primer pair amplified by nested PCR:

an outer primer pair:

the upstream primer F1: 5'-TACTTGGGATTGTGATGTG-3' the flow of the air in the air conditioner,

the downstream primer R1: 5'-AAACCACAGCACCCTTGCATGGCCA-3', respectively;

an inner primer pair:

the upstream primer F2: 5'-TAAACAGCGTCACCCAGCAG-3' the flow of the air in the air conditioner,

the downstream primer R2: 5'-CTTAGCCTGGCAAACCCTGG-3' are provided.

2. The method of claim 1, wherein the distance between the minor variation and the deletion copy number variation region in the specific DNA fragment to be verified is less than 800 bp.

3. The method of claim 1, wherein the specific DNA fragment to be verified is a DNA fragment whose presence of heterozygous minor variation and possible heterozygous deletion copy number variation is detected by high-throughput sequencing.

4. A method for validating copy number variations of a specific DNA fragment for non-disease diagnostic and therapeutic purposes, comprising the steps of: (1) using a DNA sample containing a specific DNA fragment to be verified which is known to have heterozygous micro-variation and possible heterozygous deletion copy number variation as a template, adopting a primer for verifying the CYP11B1 gene copy number variation to amplify, and carrying out sanger sequencing on an amplified product;

(2) comparing the sanger sequencing result obtained in the step (1) with a normal human control sequence; the verification results after comparison are as follows:

if the sanger sequencing result shows that the minor variation in the specific DNA segment to be verified is homozygous minor variation, the specific DNA segment to be verified has heterozygous deletion copy number variation and is positioned on a different homologous chromosome from the minor variation;

if the sanger sequencing result shows that the minor variation in the specific DNA fragment to be verified is heterozygous minor variation, the specific DNA fragment to be verified has no deletion copy number variation;

if the sanger sequencing result shows that no minor variation is detected in the specific DNA fragment to be verified, the specific DNA fragment to be verified has heterozygous deletion copy number variation and is positioned on the same homologous chromosome with the minor variation;

the specific DNA fragment to be verified is CYP11B1 gene, the heterozygous minor variation is positioned at exon 8 c.1391 — 1393dup of CYP11B1 gene, and the heterozygous deletion copy number variation possibly exists near the upstream of the heterozygous minor variation;

the primers used to verify the copy number variation of CYP11B1 gene consisted of the outside primer pair and the inside primer pair for nested PCR amplification:

an outer primer pair:

the upstream primer F1: 5'-TACTTGGGATTGTGATGTG-3' the flow of the air in the air conditioner,

the downstream primer R1: 5'-AAACCACAGCACCCTTGCATGGCCA-3', respectively;

an inner primer pair:

the upstream primer F2: 5'-TAAACAGCGTCACCCAGCAG-3' the flow of the air in the air conditioner,

the downstream primer R2: 5'-CTTAGCCTGGCAAACCCTGG-3' is added.

5. The method as claimed in claim 4, wherein the homology of the specific DNA fragment to be verified with other DNA sequences in the DNA sample is 80% or more.

6. The method as claimed in claim 5, wherein the homology of the specific DNA fragment to be verified with other DNA sequences in the DNA sample is 90% or more.

7. A system for verifying copy number variation of a specific DNA fragment, comprising

A gene amplification device, which is used for amplifying a DNA sample comprising a specific DNA fragment to be verified which is known to have heterozygous micro-variation and possibly heterozygous deletion copy number variation, wherein primers used for amplification are primers for verifying CYP11B1 gene copy number variation;

the gene sequencing device is connected with the gene amplification device and is used for carrying out sanger sequencing on the amplified product so as to obtain a sanger sequencing sequence of tiny variation in a specific DNA fragment to be verified;

the comparison device is connected with the genome sequencing device, normal human reference sequence information is prestored in the comparison device, the comparison device is used for comparing the obtained sanger sequencing sequence with the normal human reference sequence information, the tiny variation condition determined by the sanger sequencing result is obtained based on the comparison result, and then the condition of the heterozygous deletion copy number variation in the specific DNA fragment to be verified is verified by combining the known information that the heterozygous tiny variation exists in the specific DNA fragment to be verified; if the sanger sequencing result shows that the minor variation in the specific DNA segment to be verified is homozygous minor variation, the specific DNA segment to be verified has heterozygous deletion copy number variation and is positioned on a different homologous chromosome from the minor variation; if the sanger sequencing result shows that the minor variation in the specific DNA fragment to be verified is heterozygous minor variation, the specific DNA fragment to be verified has no deletion copy number variation; if the sanger sequencing result shows that no minor variation is detected in the specific DNA fragment to be verified, the specific DNA fragment to be verified has heterozygous deletion copy number variation and is positioned on the same homologous chromosome with the minor variation; and

the result output device is connected with the comparison device so as to output a verification result;

the specific DNA fragment to be verified is CYP11B1 gene, heterozygous minor variation is located at exon 8 c.1391_1393dup of CYP11B1 gene, and heterozygous deletion copy number variation possibly exists near the upstream of the heterozygous minor variation;

the primers used for verifying the copy number variation of the CYP11B1 gene consist of an outer primer pair and an inner primer pair amplified by nested PCR:

an outer primer pair:

the upstream primer F1: 5'-TACTTGGGATTGTGATGTG-3', and the adhesive tape is used for adhering the film to a substrate,

the downstream primer R1: 5'-AAACCACAGCACCCTTGCATGGCCA-3', respectively;

an inner primer pair:

the upstream primer F2: 5'-TAAACAGCGTCACCCAGCAG-3' the flow of the air in the air conditioner,

the downstream primer R2: 5'-CTTAGCCTGGCAAACCCTGG-3' is added.

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