CN109337973B

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

Info

Publication number: CN109337973B
Application number: CN201811492690.1A
Authority: CN
Inventors: 刘洪洲; 喻长顺; 熊见见; 赵薇薇; 于世辉
Original assignee: Guangzhou Kingmed Diagnostics Group Co ltd; Guangzhou Kingmed Diagnostics Central Co Ltd
Current assignee: Guangzhou Kingmed Diagnostics Group Co ltd; Guangzhou Kingmed Diagnostics Central Co Ltd
Priority date: 2018-12-07
Filing date: 2018-12-07
Publication date: 2022-06-14
Anticipated expiration: 2038-12-07
Also published as: CN109337973A

Abstract

Compared with the prior art, the primer design method has the advantages that the area for designing the primer is increased and is not limited to 80bp to 150bp, and the design difficulty of the primer is greatly reduced. The sequencing result in the verification method is very visual, and the judgment of deletion variation is more convenient. By adopting the primer design method, the verification primer, the verification method and the verification system, the influence of homology on verification can be avoided; the verification method and the verification system can determine whether the CNV exists or not and also can determine whether the CNV and the micro variation are on the same chromosome or not.

Description

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

Technical Field

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

Background

There are 46 human chromosomes, of which there are 22 pairs of autosomes, occurring in pairs, one from the father and one from the mother in each pair of chromosomes, called the homologous chromosomes. Chromosomes are mainly composed of DNA and proteins, wherein DNA, i.e., deoxyribonucleic acid sequence (also called nucleotide sequence or base sequence), determines genetic information. DNA is a long-chain polymer, and the constituent units are four kinds of deoxynucleotides, namely adenine deoxynucleotide (dAMP), thymine deoxynucleotide (dTMP), cytosine deoxynucleotide (dCMP), and guanine deoxynucleotide (dGMP). The diversity of organisms is determined by four bases in deoxynucleotide, adenine (adenine, a), thymine (thymine, T), cytosine (C) and guanine (guanine, G), the DNA sequence is usually represented by a sequence of four bases of ATCG, called base sequence or nucleotide sequence, a is paired with T, C is paired with G, and the forward direction refers to the 5 'end to 3' end direction.

Genetic variation refers to heritable variation in genomic DNA molecules, and can be classified into minor variation and Copy number variation according to the size of the fragment involved in the variation, where Copy Number Variations (CNVs) refer to deletion or repeat variations of DNA fragments greater than 1kb in length. There are various methods for detecting CNVs, such as chromosome microarray technology (CMA), single nucleotide genotyping technology, Multiplex ligation-dependent probe amplification (MLPA), and high throughput sequencing technology that has been widely used in clinical studies.

For CNVs detected like CMA, high throughput sequencing, etc., further validation is often required by other methods, commonly used methods are MLPA, semi-quantitative qPCR, etc. Among them, qPCR is one of the most commonly used methods, however, the amplification length of qPCR is required to be strict, usually 80bp to 150bp, and when the target fragment has high homology, the primer design is difficult and is very easy to fail, and further, the verification fails. MLPA is a finished reagent developed by reagent companies, and most genes have no corresponding probes. In one case, a heterozygous minor variation is detected and a heterozygous deletion CNV is detected in the vicinity, and in this case, when the homology is high, it is often difficult to verify the presence of CNV by qPCR.

Disclosure of Invention

The invention aims to provide a primer design method for verifying copy number variation of a specific DNA fragment, basically the primer design method, and also provides a primer, a kit, a method and a system for verifying copy number variation of the specific DNA fragment, so as to solve the problems in the background technology.

In order to realize the purpose, the technical scheme is as follows: a primer design method for verifying copy number variation of a specific DNA fragment, the primer design method comprising the steps of:

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

Heterozygous minor variations refer to minor variations that exist on one chromosome in the homologous chromosome, but not on the corresponding position on the other chromosome. A heterozygous deletion copy number variation is one that exists on one chromosome in the homologous chromosome and does not exist at the corresponding position on the other chromosome.

The primer of the designed DNA is a small single-stranded DNA, the upstream primer (i.e. forward primer) is complementary with the positive strand (i.e. sense strand) of the DNA, the downstream primer (i.e. reverse primer) is identical with the positive strand (i.e. sense strand) of the DNA, the primer design generally requires that the length of the primer is 15 to 30 bases, the primer can not have primer dimer and hairpin structures, and the primer needs to have specificity, i.e. a conserved region of the DNA sequence, and the like. In the invention, the method for designing the primers aiming at the specific DNA segment to be verified is that one primer in the designed primer pair is positioned at the upstream or downstream of the micro variation, and the other primer is positioned in the CNV region to be verified. Specifically, when the CNV region is located at the upstream of the microscopic variation, one primer of the designed primer pair is located at the downstream of the microscopic variation, and the other primer is located at the CNV region to be verified; when the CNV region is positioned at the downstream of the micro variation, one primer in the designed primer pair is positioned at the upstream of the micro variation, and the other primer is positioned in the CNV region to be verified; one of the advantages of such a design is that it is possible to amplify the micro-variations for the purpose of detecting them.

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 invention is the DNA fragment which is detected to have heterozygous micro-variation and possible heterozygous deletion copy number variation by the prior art. Generally, the specific DNA fragment to be verified is a DNA fragment whose presence of heterozygous minor variations and possible heterozygous deletion copy number variations is detected by high-throughput sequencing.

Preferably, the designed primer pair is two pairs, namely an outer primer pair and an inner primer pair for nested PCR amplification respectively. The influence of homology on verification can be avoided by fusing the nested PCR technology in the primer design method.

Preferably, the specific DNA fragment to be verified is CYP11B1 gene, the heterozygous minor variation is located at exon 8 c.1391 — 1393dup of CYP11B1 gene, and the heterozygous deletion copy number variation can exist near the upstream of the heterozygous minor variation.

The invention provides a primer for verifying copy number variation of a specific DNA fragment, which is designed by adopting the primer design method.

Preferably, the primers consist of an outer primer pair and an inner primer pair for nested PCR amplification:

an outer primer pair:

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

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

an inner primer pair:

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

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

The invention provides a kit for verifying copy number variation of a specific DNA fragment, which comprises the primer.

The invention provides a method for verifying copy number variation of a specific DNA fragment, which comprises the following steps:

(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 the primer designed by the primer design method to amplify, and carrying out sanger sequencing on a product obtained by amplification;

(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 located on the same homologous chromosome with the minor variation.

The Sanger method is a method in which a nucleotide starts at a certain fixed point, randomly terminates at a certain base, and is fluorescently labeled after each base to generate four sets of nucleotides of different lengths ending at A, T, C, G, which are then electrophoresed on a urea-denatured PAGE gel to be detected, thereby obtaining a visible DNA base sequence.

Preferably, the homology of the specific DNA fragment to be verified and other DNA sequences in the DNA sample is more than or equal to 80%. More preferably, the homology of the specific DNA fragment to be verified with other DNA sequences in the DNA sample is more than or equal to 90%. When the homology of the specific DNA fragment to be verified and other DNA sequences in the DNA sample is more than or equal to 80%, a proper primer is difficult to design by adopting qPCR, and particularly when the homology of the specific DNA fragment to be verified and other DNA sequences in the DNA sample is more than or equal to 90%, a proper qPCR primer is difficult to design.

The invention provides a system for verifying copy number variation of a specific DNA fragment, which comprises

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 the primer used for amplification is a primer designed by adopting the primer design method;

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

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

Has the advantages that: compared with the prior art, the primer design method has the advantages that the area of the designed primer is increased and is not limited to 80bp to 150bp, and the design difficulty of the primer is greatly reduced. The sequencing result in the verification method is very visual, and the judgment of deletion variation is more convenient. By adopting the primer design method, the verification primer, the verification method and the verification system, the influence of homology on verification can be avoided; the verification method and the verification system can determine whether the CNV exists or not and also can determine whether the CNV and the micro variation are on the same chromosome or not.

Drawings

FIG. 1 is a schematic diagram of the design of copy number variation primers for specific DNA fragments in example 1 of the present invention, in which: 1. the upstream primer F2; 2. homologous chromosome A; 3. homologous chromosome B; 4. a missing region to be verified; 5. heterozygous minor variations; 6. a downstream primer R2;

FIG. 2 is a map of the sequencing result of the upstream primer F2 in example 1, in which: 1. a normal human reference sequence; 2. a sample sequence; 3. minor variation c.1391 — 1393 dup;

FIG. 3 is a map of the sequencing result of the downstream primer R2 in example 1 of the present invention, in which: 1. a normal human reference sequence; 2. a sample sequence; 3. minor variation c.1391 — 1393 dup.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

Example 1

We detected a heterozygous deletion of exon 8 c.1391-1393 dup of the hybrid minor variant CYP11B1 gene (reference transcript NM-000497.3) on homologous chromosome A and a nucleotide sequence around upstream in the region of about 37kb at position chr8:143956640-143994311, which comprises exons 1-7 of the CYP11B1 gene, by high throughput sequencing, and then verified the heterozygous deletion fragment. Trying to design a primer by qPCR, finding that the CYP11B1 gene has a fake gene CYP11B2, the homology is very high and reaches over 90 percent, the qPCR requirement is high, the amplification length generally needs to be 80bp to 150bp, and a proper primer is difficult to design; such as: one of the primers: upstream primer 5'-TGGCAACAGGAGAGACAGGAT-3' (SEQ ID NO:5), downstream primer: 5'-GGCTCCGTATCAACCAGAGAAA-3' (SEQ ID NO:6), two primers: upstream primer 5'-TCCACCGTCCAGCTCATGT-3' (SEQ ID NO:7), downstream primer: 5'-GCCTCAAAGTGCTCCTTCCA-3' (SEQ ID NO:8), three primers: upstream primer 5'-AGGACGTGGAGAAGCTGCAA-3' (SEQ ID NO:9), downstream primer: 5'-TGCCCACGATGTTGTCTGTAG-3' (SEQ ID NO: 10). Because the primers and the amplification regions are located in the regions with homologous sequences, the quantitative amplification of the region to be detected is influenced, the deletion state cannot be correctly judged, and the qPCR experiment fails.

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

firstly, primers are designed at the downstream of the deletion region and the minor variation c.1391 — 1393dup detected by high-throughput sequencing:

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

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

And (3) carrying out long PCR by using the pair of primers and the original sample DNA detected by high-throughput sequencing as a template to obtain a product P. Wherein, the reaction system is set as table 1:

TABLE 1 first round PCR reaction System

Reagent	Volume (μ l)
		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

PCR reaction program settings are as in table 2:

TABLE 2 first round PCR reaction procedure

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

Then, primers were designed downstream of the deletion region and the minor variation c.1391 — 1393dup detected by high throughput sequencing and in the amplification region of primers F1 and R1, respectively, as shown in fig. 1:

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

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

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

Using the pair of primers, PCR amplification was performed using the long PCR product P as a template, and the reaction system was set as in table 3:

TABLE 3 second round PCR reaction System

The reaction conditions are shown in Table 4:

TABLE 4 second round PCR reaction procedure

Temperature of	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 products were subjected to sanger sequencing, the sequencing primers were the upstream primer F2 and the downstream primer R2, and the results are shown in fig. 2 and fig. 3: the sequencing results of the upstream primer F2 and the downstream primer R2, and the comparison of the sample sequence and the normal human reference sequence show that the minor variation c.1391-1393 dup is homozygous.

Finally, the results were analyzed as follows:

(1) if a minor variation c.1391 — 1393dup is detected as homozygous: CNV (chr8:143956640-143994311) is presumed to exist and to be located on a different homologous chromosome from the minor variation.

(2) If minor variation c.1391 — 1393dup is detected, it is heterozygous: it is presumed that the CNV does not exist.

(3) Minor variation c.1391 — 1393dup was not detected, i.e. only wild type was detected: CNVs are presumed to exist and to be located on the same homologous chromosome as the minor variation.

The result detected by the experiment belongs to the first condition, namely the minor variation c.1391_1393dup is homozygous, and the minor variation c.1391_1393dup is heterozygous by high-throughput sequencing, and the homologous chromosome B which is another homologous chromosome segment of the minor variation is presumed to be not amplified due to the existence of deletion, so that no sequencing data exist, and therefore, the sanger sequencing result supports the deletion variation detected by high-throughput sequencing. And the verification is successful.

In the embodiment of the invention, the long PCR amplification of the primer F1 and the primer R1 can avoid the influence of homologous sequences, and the long PCR can not be carried out if the homology is not high, for example, the homology is lower than 80 percent, and the step is omitted. The validation of the CNV by the sanger sequencing method effectively reduces the limitation of PCR primer design, and simultaneously, whether adjacent micro-variation and CNV are on the same chromosome can be presumed, which may have certain significance for the explanation of autosomal recessive genetic diseases.

In comparison, qPCR has high design requirements, short amplification length, strict requirements, high primer design difficulty and relatively poor experimental stability. When the homology is high, it is often difficult to design a proper primer, so that it is difficult to perform a verification experiment. Because qPCR is a semi-quantitative detection for the amplification of a region to be detected, the relative position of a deletion region and a minor variation, namely whether the deletion region and the minor variation are on the same homologous chromosome, cannot be known.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

<110> Guangzhou gold-area medical testing center, Inc., Guangzhou gold-area medical testing group, Inc

<120> primer design method, primer, kit and method for verifying copy number variation of specific DNA fragment

Method and system

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 19

<212> DNA

<213> Artificial sequence

<400> 1

tacttgggat tgtgatgtg 19

<210> 2

<211> 25

<212> DNA

<213> Artificial sequence

<400> 2

aaaccacagc acccttgcat ggcca 25

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<400> 3

taaacagcgt cacccagcag 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<400> 4

cttagcctgg caaaccctgg 20

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<400> 5

tggcaacagg agagacagga t 21

<210> 6

<211> 22

<212> DNA

<213> Artificial sequence

<400> 6

ggctccgtat caaccagaga aa 22

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence

<400> 7

tccaccgtcc agctcatgt 19

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<400> 8

gcctcaaagt gctccttcca 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<400> 9

aggacgtgga gaagctgcaa 20

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence

<400> 10

tgcccacgat gttgtctgta g 21

Claims

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;

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

an inner primer pair:

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