CN112375818A - Detection method of SNP (single nucleotide polymorphism) locus of SMA (shape memory alloy) gene - Google Patents
Detection method of SNP (single nucleotide polymorphism) locus of SMA (shape memory alloy) gene Download PDFInfo
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Abstract
The invention discloses a detection method of SNP locus of SMA gene, which comprises the steps of preparing a glass sheet, adding a protective group on the glass substrate, carrying out first photoetching, second photoetching and third photoetching to add a first base on the glass substrate, connecting the first base to the glass substrate, paving a second base on the glass substrate, removing the protective group in the second round of light irradiation to grow a second base, adding a third base, increasing the third base for growing a DNA chain on the glass substrate, marking by a fluorescent chemical substance, sequentially injecting a gene to be detected onto the glass substrate embedded with a chip, and carrying out laser scanning according to the variation of a detected sequence at the fluorescent intensity detection position. The detection method of the SNP locus of the SMA gene solves the problems of small detection flux, low detection accuracy and high cost.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a method for detecting SNP loci of SMA genes.
Background
Spinal Muscular Atrophy (SMA) is a disease in which motor neurons at the anterior horn of the spinal cord degenerate, causing muscle weakness and atrophy. Belongs to autosomal recessive genetic diseases, and is not uncommon clinically. The clinical manifestations of this disease are very different, and according to the onset age and clinical course of disease, SMA is classified into type 4 from heavy to light. The common characteristics are degeneration of anterior angle cells of spinal cord, progressive and symmetrical clinical manifestations, extensive flaccid paralysis and muscular atrophy with the main limb proximal, and normal intelligence development and feeling, wherein Single Nucleotide Polymorphism (SNP) refers to DNA sequence polymorphism caused by variation of single nucleotide on genome level. It is the most common one of the human heritable variations, accounting for over 90% of all known polymorphisms. SNPs are widely present in the human genome, averaging 1 per 300 base pairs, and the total number is estimated to be 300 ten thousand or more. A SNP is a two-state marker, caused by a transition or transversion of a single base, or by an insertion or deletion of a base. SNPs may be in either the gene sequence or non-coding sequences outside the gene.
The existing sequencing method is a quantitative polymerase chain reaction or a direct sequencing method, and has the following problems;
firstly, the detection flux is smaller;
secondly, the detection accuracy is not high;
thirdly, the cost is expensive.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a detection method of the SNP locus of the SMA gene, which solves the problems of small detection flux, low detection accuracy and high cost.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a detection method of SNP locus of SMA gene comprises the following steps:
s1, preparing a glass substrate, adding a protecting group on the glass substrate, and preventing the following DNA extension reaction through the protecting group;
s2, next, carrying out first photoetching, namely, arranging small squares on a glass substrate, covering a part of glass plate area by using a photomask to prevent ultraviolet light from irradiating the glass substrate, and irradiating the ultraviolet light on the corresponding places of the small squares through the local shielding of the photomask to ensure that protective groups in the small squares can be irradiated by the ultraviolet light, wherein photosensitive protective groups fall off from the original connected hydroxyl groups, and the protective groups are not shielded by the photomask and are still connected to the original hydroxyl groups because of not being irradiated by the light;
s3, next, applying a substrate of the first base to be linked to a glass substrate, wherein the substrate has been irradiated with light and has been removed of the protecting group, so that the substrate binds to the first base, and the substrate is not bound to the first base when covered by the opaque region of the mask and the protecting group is left;
s4, carrying out the second photo-etching, namely, taking a second photo-mask cover to cover the glass substrate, irradiating the glass substrate by ultraviolet light through the second photo-mask again, after the second light irradiation, removing the protective group on the glass substrate corresponding to the transparent part of the second photo-mask, then spreading the second base of the second round of planting on the glass substrate, wherein the position of the glass substrate, from which the protective group is removed in the second round of light irradiation, can grow a second base;
s5, covering with a third mask, irradiating with light for the third time, adding a third base, repeating the process, and sequentially arranging different positions on the glass substrate to grow the desired DNA chain;
s6, extracting the gene to be detected, slicing the gene to be detected in different lengths, marking the gene by a fluorescent chemical substance, and sequentially injecting the gene to be detected onto a glass substrate embedded with a chip;
since the degree of hybridization of S7 and the DNA strand is correlated with the fluorescence intensity, the variation of the sequence to be detected at the site of detection may be detected by the intensity of fluorescence by laser scanning.
Preferably, the glass substrate in the step S1 is composed of any one or more of glass, silicon and polypropylene.
Preferably, the small squares of the glass substrate in the step S1 have one of the shapes of 3 × 6, 4 × 8 and 6 × 12.
Preferably, the surface of the photomask in steps S2, S4 and S5 is in the form of small squares, and the small squares are in one of 3 × 6, 4 × 8 and 6 × 12, and the small squares on the surface of the photomask are respectively made of transparent and opaque materials, and are distributed and sorted differently.
Preferably, the small grids on the surface of the photomask and the small grids on the surface of the glass sheet are matched with each other, and the small grids on the surface of the photomask and the surface of the glass sheet are both in a micron order.
Preferably, the DNA strand is a probe having a specific base sequence.
Preferably, the fluorescent chemical in the S6 step is composed of any one or more of dinitrobenzene, biotin and polymerase.
Preferably, the length of the probe is 30-50 UM.
(III) advantageous effects
The invention provides a detection method of SNP loci of SMA genes. Compared with the prior art, the method has the following beneficial effects:
1. the detection method of the SNP locus of the SMA gene comprises the steps of preparing a glass sheet, adding a protective group on the glass substrate, carrying out first photoetching, second photoetching and third photoetching to enable the glass substrate to grow a DNA chain, marking the glass substrate by a fluorescent chemical substance, sequentially injecting genes to be detected onto the glass substrate embedded with a chip, and scanning by laser.
Drawings
FIG. 1 is a flow chart of the detection method of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment of the invention provides a technical scheme that: a detection method of SNP locus of SMA gene comprises the following steps:
s1, preparing a glass substrate, adding a protecting group on the glass substrate, and preventing the following DNA extension reaction through the protecting group;
s2, next, carrying out first photoetching, namely, arranging small squares on a glass substrate, covering a part of glass plate area by using a photomask to prevent ultraviolet light from irradiating the glass substrate, and irradiating the ultraviolet light on the corresponding places of the small squares through the local shielding of the photomask to ensure that protective groups in the small squares can be irradiated by the ultraviolet light, wherein photosensitive protective groups fall off from the original connected hydroxyl groups, and the protective groups are not shielded by the photomask and are still connected to the original hydroxyl groups because of not being irradiated by the light;
s3, next, applying a substrate of the first base to be linked to a glass substrate, wherein the substrate has been irradiated with light and has been removed of the protecting group, so that the substrate binds to the first base, and the substrate is not bound to the first base when covered by the opaque region of the mask and the protecting group is left;
s4, carrying out the second photo-etching, namely, taking a second photo-mask cover to cover the glass substrate, irradiating the glass substrate by ultraviolet light through the second photo-mask again, after the second light irradiation, removing the protective group on the glass substrate corresponding to the transparent part of the second photo-mask, then spreading the second base of the second round of planting on the glass substrate, wherein the position of the glass substrate, from which the protective group is removed in the second round of light irradiation, can grow a second base;
s5, covering with a third mask, irradiating with light for the third time, adding a third base, repeating the process, and sequentially arranging different positions on the glass substrate to grow the desired DNA chain;
s6, extracting the gene to be detected, slicing the gene to be detected in different lengths, marking the gene by a fluorescent chemical substance, and sequentially injecting the gene to be detected onto a glass substrate embedded with a chip;
since the degree of hybridization of S7 and the DNA strand is correlated with the fluorescence intensity, the variation of the sequence to be detected at the site of detection may be detected by the intensity of fluorescence by laser scanning.
Further, the glass substrate in the step S1 is composed of glass and silicon.
Further, the small squares of the glass substrate in the step S1 have a shape of 3 × 6.
Further, the surfaces of the masks in the steps S2, S4 and S5 are small squares, the small squares are 3 × 6 in shape, and the small squares on the surfaces of the masks are respectively made of transparent and opaque materials, and are all in different distribution and ordering.
Furthermore, the small grids on the surface of the photomask and the small grids on the surface of the glass sheet are matched with each other, and the small grids on the surface of the photomask and the surface of the glass sheet are both in a micron order.
Further, the DNA strand is a probe having a specific base sequence.
Further, the fluorescent chemical in step S6 is composed of one of dinitrobenzenes.
Further, the length of the probe was 30 UM.
Example 2
The embodiment of the invention provides a technical scheme that: a detection method of SNP locus of SMA gene comprises the following steps:
s1, preparing a glass substrate, adding a protecting group on the glass substrate, and preventing the following DNA extension reaction through the protecting group;
s2, next, carrying out first photoetching, namely, arranging small squares on a glass substrate, covering a part of glass plate area by using a photomask to prevent ultraviolet light from irradiating the glass substrate, and irradiating the ultraviolet light on the corresponding places of the small squares through the local shielding of the photomask to ensure that protective groups in the small squares can be irradiated by the ultraviolet light, wherein photosensitive protective groups fall off from the original connected hydroxyl groups, and the protective groups are not shielded by the photomask and are still connected to the original hydroxyl groups because of not being irradiated by the light;
s3, next, applying a substrate of the first base to be linked to a glass substrate, wherein the substrate has been irradiated with light and has been removed of the protecting group, so that the substrate binds to the first base, and the substrate is not bound to the first base when covered by the opaque region of the mask and the protecting group is left;
s4, carrying out the second photo-etching, namely, taking a second photo-mask cover to cover the glass substrate, irradiating the glass substrate by ultraviolet light through the second photo-mask again, after the second light irradiation, removing the protective group on the glass substrate corresponding to the transparent part of the second photo-mask, then spreading the second base of the second round of planting on the glass substrate, wherein the position of the glass substrate, from which the protective group is removed in the second round of light irradiation, can grow a second base;
s5, covering with a third mask, irradiating with light for the third time, adding a third base, repeating the process, and sequentially arranging different positions on the glass substrate to grow the desired DNA chain;
s6, extracting the gene to be detected, slicing the gene to be detected in different lengths, marking the gene by a fluorescent chemical substance, and sequentially injecting the gene to be detected onto a glass substrate embedded with a chip;
since the degree of hybridization of S7 and the DNA strand is correlated with the fluorescence intensity, the variation of the sequence to be detected at the site of detection may be detected by the intensity of fluorescence by laser scanning.
Further, the glass substrate in the step S1 is composed of glass and polypropylene.
Further, the small squares of the glass substrate in the S1 step have a shape of 4 × 8.
Furthermore, the surfaces of the masks in the steps S2, S4 and S5 are small squares, the small squares are 4 × 8 in shape, and the small squares on the surfaces of the masks are respectively made of transparent and opaque materials, and are all in different distribution and ordering.
Furthermore, the small grids on the surface of the photomask and the small grids on the surface of the glass sheet are matched with each other, and the small grids on the surface of the photomask and the surface of the glass sheet are both in a micron order.
Further, the DNA strand is a probe having a specific base sequence.
Further, the fluorescent chemical in the step S6 is composed of any one or more of dinitrobenzene, biotin and polymerase.
Further, the length of the probe was 40 UM.
Example 3
The embodiment of the invention provides a technical scheme that: a detection method of SNP locus of SMA gene comprises the following steps:
s1, preparing a glass substrate, adding a protecting group on the glass substrate, and preventing the following DNA extension reaction through the protecting group;
s2, next, carrying out first photoetching, namely, arranging small squares on a glass substrate, covering a part of glass plate area by using a photomask to prevent ultraviolet light from irradiating the glass substrate, and irradiating the ultraviolet light on the corresponding places of the small squares through the local shielding of the photomask to ensure that protective groups in the small squares can be irradiated by the ultraviolet light, wherein photosensitive protective groups fall off from the original connected hydroxyl groups, and the protective groups are not shielded by the photomask and are still connected to the original hydroxyl groups because of not being irradiated by the light;
s3, next, applying a substrate of the first base to be linked to a glass substrate, wherein the substrate has been irradiated with light and has been removed of the protecting group, so that the substrate binds to the first base, and the substrate is not bound to the first base when covered by the opaque region of the mask and the protecting group is left;
s4, carrying out the second photo-etching, namely, taking a second photo-mask cover to cover the glass substrate, irradiating the glass substrate by ultraviolet light through the second photo-mask again, after the second light irradiation, removing the protective group on the glass substrate corresponding to the transparent part of the second photo-mask, then spreading the second base of the second round of planting on the glass substrate, wherein the position of the glass substrate, from which the protective group is removed in the second round of light irradiation, can grow a second base;
s5, covering with a third mask, irradiating with light for the third time, adding a third base, repeating the process, and sequentially arranging different positions on the glass substrate to grow the desired DNA chain;
s6, extracting the gene to be detected, slicing the gene to be detected in different lengths, marking the gene by a fluorescent chemical substance, and sequentially injecting the gene to be detected onto a glass substrate embedded with a chip;
since the degree of hybridization of S7 and the DNA strand is correlated with the fluorescence intensity, the variation of the sequence to be detected at the site of detection may be detected by the intensity of fluorescence by laser scanning.
Further, the glass substrate in the step S1 is composed of glass, silicon, and polypropylene.
Further, the small squares of the glass substrate in the S1 step have a 6 × 12 shape.
Further, the surfaces of the masks in steps S2, S4 and S5 are squares, the squares are 6 × 12 in shape, and the squares on the surfaces of the masks are respectively made of transparent and opaque materials, and are all distributed and sorted differently.
Furthermore, the small grids on the surface of the photomask and the small grids on the surface of the glass sheet are matched with each other, and the small grids on the surface of the photomask and the surface of the glass sheet are both in a micron order.
Further, the DNA strand is a probe having a specific base sequence.
Further, the fluorescent chemical in the step S6 is composed of any one or more of dinitrobenzene, biotin and polymerase.
Further, the length of the probe was 50 UM.
In this example, it is noted that in organic synthesis, molecules having 2 or more functional groups include most of basic groups in a basic organic substance containing a nitrogen atom, which are called nitrogenous bases, in order to prevent a functional group from being destroyed by reaction, and amino groups (-NH2) are the simplest nitrogenous bases, which are also called nucleobases and nitrogenous bases in biochemistry, and are nitrogen-containing compounds that form nucleosides.
And those not described in detail in this specification are well within the skill of those in the art.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A detection method of SNP locus of SMA gene is characterized in that: the detection method comprises the following steps:
s1, preparing a glass substrate, adding a protecting group on the glass substrate, and preventing the following DNA extension reaction through the protecting group;
s2, next, carrying out first photoetching, namely, arranging small squares on a glass substrate, covering a part of glass plate area by using a photomask to prevent ultraviolet light from irradiating the glass substrate, and irradiating the ultraviolet light on the corresponding places of the small squares through the local shielding of the photomask to ensure that protective groups in the small squares can be irradiated by the ultraviolet light, wherein photosensitive protective groups fall off from the original connected hydroxyl groups, and the protective groups are not shielded by the photomask and are still connected to the original hydroxyl groups because of not being irradiated by the light;
s3, next, applying a substrate of the first base to be linked to a glass substrate, wherein the substrate has been irradiated with light and has been removed of the protecting group, so that the substrate binds to the first base, and the substrate is not bound to the first base when covered by the opaque region of the mask and the protecting group is left;
s4, carrying out the second photo-etching, namely, taking a second photo-mask cover to cover the glass substrate, irradiating the glass substrate by ultraviolet light through the second photo-mask again, after the second light irradiation, removing the protective group on the glass substrate corresponding to the transparent part of the second photo-mask, then spreading the second base of the second round of planting on the glass substrate, wherein the position of the glass substrate, from which the protective group is removed in the second round of light irradiation, can grow a second base;
s5, covering with a third mask, irradiating with light for the third time, adding a third base, repeating the process, and sequentially arranging different positions on the glass substrate to grow the desired DNA chain;
s6, extracting the gene to be detected, slicing the gene to be detected in different lengths, marking the gene by a fluorescent chemical substance, and sequentially injecting the gene to be detected onto a glass substrate embedded with a chip;
since the degree of hybridization of S7 and the DNA strand is correlated with the fluorescence intensity, the variation of the sequence to be detected at the site of detection may be detected by the intensity of fluorescence by laser scanning.
2. The method for detecting SNP sites of SMA gene according to claim 1, wherein: the glass substrate in the step S1 is made of any one or more of glass, silicon and polypropylene.
3. The method for detecting SNP sites of SMA gene according to claim 1, wherein: the small squares of the glass substrate in the step S1 are in one of the shapes of 3 × 6, 4 × 8, and 6 × 12.
4. The method for detecting SNP sites of SMA gene according to claim 1, wherein: the surfaces of the masks in the steps S2, S4 and S5 are small squares, the small squares are in one of the shapes of 3 × 6, 4 × 8 and 6 × 12, the small squares on the surfaces of the masks are respectively made of transparent and opaque, and the distribution and the ordering are different.
5. The method for detecting SNP site of SMA gene according to claim 4, wherein: the small grids on the surface of the photomask and the small grids on the surface of the glass sheet are matched with each other, and the small grids on the surface of the photomask and the surface of the glass sheet are both in a micron order.
6. The method for detecting SNP sites of SMA gene according to claim 1, wherein: the DNA strand is a probe having a specific base sequence.
7. The method for detecting SNP sites of SMA gene according to claim 1, wherein: the fluorescent chemical in the step S6 is composed of any one or more of dinitrobenzene, biotin, and polymerase.
8. The method for detecting SNP site of SMA gene according to claim 6, wherein: the length of the probe is 30-50 UM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011285874.8A CN112375818A (en) | 2020-11-17 | 2020-11-17 | Detection method of SNP (single nucleotide polymorphism) locus of SMA (shape memory alloy) gene |
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| CN111073884A (en) * | 2020-02-14 | 2020-04-28 | 昆明理工大学 | Method for improving accuracy of SNP locus detection with functional effect in SNP with distance of non-coding region less than 50bp |
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