CN110699464A - A Variety Screening Method Based on Molecular Genetic Marker Technology - Google Patents

Abstract

The invention discloses a variety screening method based on molecular genetic marker technology, belonging to the technical field of biological genetic testing. A variety screening method based on molecular genetic marker technology includes the following steps: extracting DNA, collecting liver from a target object Tissue 5-10g, put it into a DNA high-efficiency separation tube, extract genomic DNA, PCR amplification, mix double-distilled water, PCR buffer, dNTP, PCR primers, TaqDNA polymerase and genomic DNA in turn, and then in The cyclic reaction is carried out on the PCR amplifier, and the PCR product is purified after the reaction. The present invention uses the DNA high-efficiency separation tube to extract the target DNA, which optimizes the DNA extraction process, improves the DNA extraction efficiency, and also obtains higher The purity of the target DNA, on the premise of improving the purity of the target DNA, further improves the accuracy of the analysis results of the molecular genetic marker technology, so that the desired DNA type can be selected more accurately.

Description

A Variety Screening Method Based on Molecular Genetic Marker Technology

technical field

The present invention relates to the technical field of biological genetic testing, and more particularly, to a method for screening varieties based on molecular genetic marker technology.

Background technique

Molecular genetic markers are based on DNA fragment length polymorphisms caused by species mutation, and have many advantages. First, they can directly detect differences in DNA levels and are not limited by time and space. Second, the number of markers is abundant and the polymorphism is high. 3. Co-dominant markers can distinguish between homozygotes and heterozygotes; 4. It can explain the genetic variation of some individuals in the family; 5. It can identify individuals of different genders and ages.

The widely used molecular genetic markers include: RFLP analysis technology, DNA fingerprint analysis technology, RAPD analysis technology and AFLP analysis technology.

With the development of genetic engineering, especially DNA recombination technology, it is now known that animals not only have polymorphisms in coat color, body shape, blood type, chromosomes, etc., but also polymorphisms at the DNA level, especially after the 1980s. Various genetic marker methods for studying DNA polymorphisms have developed extremely rapidly, and the application of molecular genetic markers in animal breeding has become a reality. Predicting the heterosis between combinations based on the heterozygosity of the molecular marker loci can greatly reduce the work of combining ability determination. The identification and selection of hybrid progeny is an important part of breeding work.

In molecular genetic marker technology, the process of DNA extraction is extremely important, which seriously affects the accuracy of analysis results. More complicated, and the DNA extraction purity is poor.

SUMMARY OF THE INVENTION

1. Technical problems to be solved

In view of the problems existing in the prior art, the purpose of the present invention is to provide a variety screening method based on molecular genetic marker technology, which optimizes the DNA extraction process by using a DNA high-efficiency separation tube to extract the target DNA, and improves the DNA extraction process. At the same time of efficiency, high-purity target DNA is also obtained. Under the premise of improving the purity of target DNA, the accuracy of the analysis results of molecular genetic marker technology has been further improved, so that the desired target DNA can be selected more accurately. DNA type.

2. Technical solutions

In order to solve the above problems, the present invention adopts the following technical solutions.

A variety screening method based on molecular genetic marker technology, comprising the following steps:

Step 1: Extract DNA, collect 5-10g of liver tissue from the target object, and put it into a DNA high-efficiency separation tube to extract genomic DNA;

Step 2: PCR amplification, mix double-distilled water, PCR buffer, dNTP, PCR primers, TaqDNA polymerase and genomic DNA in turn, and then perform a cyclic reaction on a PCR amplifier, and purify the PCR product after the reaction;

Step 3: Enzymatic cleavage, mixing sterile water, buffer, BSA, restriction endonuclease and the purified PCR product, and analyzing the enzymatic cleavage product by agarose gel electrophoresis;

Step 4: Hybridization analysis, DNA fragments are transferred to a support membrane (nylon membrane or nitrocellulose membrane) by Southern blotting, and then probes labeled with radioisotopes (32P) or non-isotopes (such as digoxigenin, fluorescein) are used Hybridize with the DNA fragments on the support membrane, and finally do autoradiography to detect polymorphic bands and identify DNA differences.

The invention optimizes the DNA extraction process by using a DNA high-efficiency separation tube to extract the target DNA, improves the DNA extraction efficiency, and also obtains the target DNA of higher purity, and under the premise of improving the purity of the target DNA, the molecular The accuracy of the analysis results of genetic marker technology has been further improved, so that the desired DNA type can be selected more accurately.

Further, the DNA high-efficiency separation tube includes an inner tube and an open sleeve, the open sleeve is sleeved on the outer side of the inner tube, the inner wall of the open sleeve is fixedly connected with a sealing sleeve, and the inner tube is provided with a row. The liquid tank and the open sleeve can slide up and down along the outer wall of the inner tube, the drain groove is used for the outflow of the supernatant, and the sealing sleeve is used to improve the sealing performance, so that the liquid in the inner tube is not easy to flow into the gap between the inner tube and the open sleeve.

Further, the open end of the open sleeve is fixedly connected with a pair of main limit plates, the outer end of the inner tube is fixedly connected with a secondary limit plate and a baffle plate, and the auxiliary limit plate is fixedly connected to the baffle plate. At the lower end, the auxiliary limit plate is slidably connected between a pair of main limit plates, bolts are connected between the main limit plate and the auxiliary limit plate, and the main limit plate and the auxiliary limit plate are used in cooperation , the opening sleeve can be limited, so that the opening sleeve is not easy to rotate while sliding up and down.

Further, both the main limit plate and the auxiliary limit plate are provided with connecting holes, and the bolts are threadedly connected to the inside of the connecting holes. The auxiliary limit plate, thereby fixing the open sleeve.

Further, the width of the baffle plate is larger than the width of the auxiliary limit plate, and the opening sleeve is conveniently positioned by the baffle plate. When fixing the opening sleeve, the opening sleeve is moved upward until the main limit plate contacts the lower end of the baffle plate. , at this time, the connecting hole on the main limit plate is connected with the connecting hole on the auxiliary limit plate, which is convenient for bolt fixing.

Further, a transfer frame is provided on the open sleeve, the transfer frame includes a U-shaped frame, a limit groove is defined on the upper end of the open sleeve, the U-shaped frame matches the limit groove, and the transfer frame is used for the transfer frame. to drain the supernatant.

Further, a pair of side ends of the U-shaped frame are fixedly connected with a main arc-shaped piece and an auxiliary arc-shaped piece, the main arc-shaped piece is located on one side of the auxiliary arc-shaped piece, and the main arc-shaped piece and the auxiliary arc-shaped piece are fixedly connected. The plate facilitates the positioning of the U-shaped frame so that it can be stably positioned on the inside of the drain and limit grooves.

Further, a pair of side ends of the U-shaped frame is also fixedly connected with a sealing gasket, the sealing gasket is located between the main arc-shaped sheet and the secondary arc-shaped sheet, and the sealing gasket is used to improve the sealing performance. During the process, the liquid is not easy to flow out along the outer side wall of the U-shaped frame.

Further, the width of the notch of the drain groove is the same as the width of the notch of the limiting groove.

Further, a sealing cover is also provided on the upper side of the inner tube, and the sealing cover is clamped to the outer end of the open sleeve.

3. Beneficial effects

Compared with the prior art, the advantages of the present invention are:

(1) This scheme optimizes the DNA extraction process by using a DNA high-efficiency separation tube to extract the target DNA. While improving the DNA extraction efficiency, it also obtains a higher-purity target DNA. On the premise of improving the purity of the target DNA , so that the accuracy of the analysis results of molecular genetic marker technology has been further improved, so that the required DNA type can be selected more accurately.

(2) The DNA high-efficiency separation tube includes an inner tube and an open sleeve. The open sleeve is sleeved on the outside of the inner tube. The inner wall of the open sleeve is fixedly connected with a sealing sleeve. The outer wall of the inner tube slides up and down, the drain groove is used for the outflow of the supernatant, and the sealing sleeve is used to improve the sealing, so that the liquid in the inner tube is not easy to flow into the gap between the inner tube and the open sleeve.

(3) A pair of main limit plates are fixedly connected to the open end of the open sleeve, the outer end of the inner tube is fixedly connected with a sub limit plate and a baffle, the auxiliary limit plate is fixedly connected to the lower end of the baffle, and the auxiliary limit plate It is slidably connected between a pair of main limit plates, and bolts are connected between the main limit plate and the auxiliary limit plate. It is difficult to rotate the open sleeve while sliding up and down.

(4) Both the main limit plate and the auxiliary limit plate are provided with connecting holes, and the bolts are connected to the inside of the connecting holes. , thereby fixing the split sleeve.

(5) The width of the baffle is larger than the width of the auxiliary limit plate, and the opening sleeve is conveniently positioned by the baffle. When fixing the opening sleeve, move the opening sleeve upward until the main limit plate contacts the lower end of the baffle. When the connecting hole on the main limit plate is communicated with the connecting hole on the auxiliary limit plate, it is convenient to fix the bolts.

(6) There is a transfer frame on the open sleeve, the transfer frame includes a U-shaped frame, a limit groove is set on the upper end of the open sleeve, the U-shaped frame matches the limit groove, and the transfer frame is used to discharge the supernatant.

(7) A pair of side ends of the U-shaped frame are fixedly connected with the main arc-shaped piece and the auxiliary arc-shaped piece. The main arc-shaped piece is located on one side of the auxiliary arc-shaped piece. The frame is positioned so that it can be stably positioned inside the drain groove and the limit groove.

(8) A pair of side ends of the U-shaped frame is also fixedly connected with a sealing gasket. The sealing gasket is located between the main arc-shaped sheet and the secondary arc-shaped sheet. The sealing gasket is used to improve the sealing performance. In the process of discharging the supernatant, So that the liquid is not easy to flow out along the outer wall of the U-shaped frame.

Description of drawings

Fig. 1 is the perspective view one of the DNA high-efficiency separation tube of the present invention;

Fig. 2 is the perspective view two of the DNA high-efficiency separation tube of the present invention;

Fig. 3 is the perspective view three of the DNA high-efficiency separation tube of the present invention;

4 is a perspective view of the inner tube and the open sleeve of the present invention before installation;

Fig. 5 is the perspective view of the transfer frame of the present invention;

6 is a perspective view four of the DNA high-efficiency separation tube of the present invention;

FIG. 7 is a perspective view 5 of the high-efficiency DNA separation tube of the present invention.

Description of the labels in the figure:

1 inner pipe, 101 drain groove, 2 open sleeve, 201 limit groove, 3 sealing sleeve, 4 main limit plate, 5 auxiliary limit plate, 6 baffle plate, 7 connecting holes, 8 bolts, 9 transfer frame, 901 U-shaped frame, 902 main arc-shaped sheet, 903 secondary arc-shaped sheet, 904 sealing gasket, 10 sealing cover.

Detailed ways

The following will combine the drawings in the embodiments of the present invention; the technical solutions in the embodiments of the present invention will be described clearly and completely; obviously; the described embodiments are only a part of the embodiments of the present invention; rather than all the embodiments, based on The embodiments of the present invention; all other embodiments obtained by those of ordinary skill in the art without creative work; all belong to the protection scope of the present invention.

In the description of the present invention, it should be noted that the orientations or positional relationships indicated by the terms "upper", "lower", "inner", "outer", "top/bottom", etc. are based on the orientations shown in the accompanying drawings Or the positional relationship is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed to indicate or imply relative importance.

In the description of the present invention, it should be noted that, unless otherwise expressly specified and limited, the terms "installation", "provided with", "sleeve/connection", "connection", etc., should be understood in a broad sense, such as " Connection", which can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, and it can be an internal connection between two components. of connectivity. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood in specific situations.

Example:

A variety screening method based on molecular genetic marker technology, comprising the following steps:

Step 1: Extract DNA, collect 5-10g of liver tissue from the target object, and put it into a DNA high-efficiency separation tube to extract genomic DNA;

Step 2: PCR amplification, mix double-distilled water, PCR buffer, dNTP, PCR primers, TaqDNA polymerase and genomic DNA in turn, and then perform a cyclic reaction on a PCR amplifier, and purify the PCR product after the reaction;

Step 3: Enzymatic cleavage, mixing sterile water, buffer, BSA, restriction endonuclease and the purified PCR product, and analyzing the enzymatic cleavage product by agarose gel electrophoresis;

Step 4: Hybridization analysis, transfer the DNA fragments to the support membrane nylon membrane or nitrocellulose membrane by Southern blot, and then use radioisotope 32P or non-isotope such as digoxigenin, fluorescein-labeled probe and DNA on the support membrane The fragments are hybridized, and finally autoradiographic, polymorphic bands are detected, and DNA differences are identified.

Please refer to FIG. 1, the DNA high-efficiency separation tube includes an inner tube 1 and an open sleeve 2, the open sleeve 2 is sleeved on the outer side of the inner tube 1, the open sleeve 2 can slide up and down along the outer wall of the inner tube 1, and the inner tube 1 is opened There is a drain groove 101. The drain tank 101 is used for the outflow of the supernatant. Please refer to FIG. 4. A sealing sleeve 3 is fixedly connected to the inner wall of the open sleeve 2. The sealing sleeve 3 is used to improve the sealing performance, so that the liquid in the inner tube 1 It is difficult to flow into the gap between the inner tube 1 and the open sleeve 2 .

Please refer to FIG. 4 , the open end of the split sleeve 2 is fixedly connected with a pair of main limit plates 4 , the outer end of the inner tube 1 is fixedly connected with a secondary limit plate 5 and a baffle 6 , and the auxiliary limit plate 5 is fixedly connected to the stopper At the lower end of the plate 6, the auxiliary limit plate 5 is slidably connected between a pair of main limit plates 4. Through the cooperation of the main limit plate 4 and the auxiliary limit plate 5, the opening sleeve 2 can be limited, so that the opening sleeve 2 can be limited. The open sleeve 2 is not easy to rotate while sliding up and down.

Please refer to FIG. 2 , bolts 8 are connected between the main limit plate 4 and the auxiliary limit plate 5 , please refer to FIGS. 3 and 4 , the main limit plate 4 and the auxiliary limit plate 5 are provided with connecting holes 7 The bolts 8 are threadedly connected to the inside of the connecting holes 7. When the supernatant is not required to be discharged, the main limiting plate 4 and the secondary limiting plate 5 are connected and fixed by the bolts 8, thereby fixing the open sleeve 2.

Please refer to Fig. 1, the width of the baffle 6 is larger than the width of the auxiliary limit plate 5, the opening sleeve 2 is conveniently positioned by the baffle 6, when the opening sleeve 2 is fixed, the opening sleeve 2 is moved up until the main limit plate 4 is in contact with the lower end of the baffle plate 6. At this time, the connecting holes 7 on the main limiting plate 4 communicate with the connecting holes 7 on the auxiliary limiting plate 5, which facilitates the fixing of the bolts 8.

Please refer to FIG. 5 and FIG. 6 , the open sleeve 2 is provided with a transfer frame 9 , the transfer frame 9 includes a U-shaped frame 901 , a limiting groove 201 is defined on the upper end of the open sleeve 2 , the slot width and limit of the drain groove 101 are The slot width of the groove 201 is the same, the U-shaped frame 901 is matched with the limiting groove 201, the transfer frame 9 is used to discharge the supernatant, and a pair of side ends of the U-shaped frame 901 are fixedly connected with the main arc-shaped sheet 902 and the auxiliary The arc-shaped piece 903, the main arc-shaped piece 902 is located on one side of the auxiliary arc-shaped piece 903, the main arc-shaped piece 902 and the auxiliary arc-shaped piece 903 facilitate the positioning of the U-shaped frame 901.

When in use, the main arc-shaped sheet 902 is closely attached to the inner wall of the inner tube 1, and the secondary arc-shaped sheet 903 is closely attached to the outer wall of the open sleeve 2, so that the U-shaped frame 901 can be stably located between the drain groove 101 and the limit groove 201. On the inner side, a pair of side ends of the U-shaped frame 901 is also fixedly connected with a sealing gasket 904. The sealing gasket 904 is located between the main arc-shaped sheet 902 and the secondary arc-shaped sheet 903. The sealing gasket 904 is used to improve the sealing performance. During the process of removing the liquid, the liquid is not easy to flow out along the outer side wall of the U-shaped frame 901 .

Referring to FIG. 7 , the upper side of the inner tube 1 is further provided with a sealing cover 10 , and the sealing cover 10 is clamped to the outer end of the open sleeve 2 .

In step 1, the method for extracting DNA through a DNA high-efficiency separation tube is as follows:

S1. Put 8g of liver tissue into the inner tube 1, add 16ml of 0.1mol/L sodium chloride-0.05mol/L sodium citrate mixture, fully grind to obtain a homogenate;

S2, the homogenate is centrifuged to obtain a supernatant and a precipitate, and the precipitate is taken;

S3. Add 15 mL of 1 mol/L NaCl solution to the precipitation, mix well and centrifuge, and take the supernatant;

S4, add an equal volume of chloroform-isoamyl alcohol to the supernatant, shake vigorously and then centrifuge, and take the supernatant;

S5. An equal volume of 95% ethanol was added to the supernatant, and white filamentous precipitates were seen, and then slowly wound with a glass rod to take out the precipitate, which was DNA.

Generally, in the process of DNA extraction, it is necessary to take the supernatant or the precipitation several times for reaction, that is, the precipitation and the supernatant need to be separated. The traditional method is to suck the supernatant through a capillary pipette. The operation steps are: first Pinch the rubber nipple, then insert it into the solution (if the rubber nipple is squeezed after inserting the solution, it will muddy the clear liquid), and then slowly loosen the rubber nipple, so that the solution is slowly sucked into the tube, so that the precipitate and the solution Separation, this method is not only time-consuming and inefficient, but also easily muddies the supernatant during the aspiration process, resulting in lower purity of the extracted DNA.

In the process of DNA extraction by the DNA high-efficiency separation tube of the present invention, when the precipitate and the supernatant need to be separated, the bolt 8 only needs to be taken out, and the transfer frame 9 is placed inside the drain groove 101 and the limit groove 201, as shown in FIG. 6 . As shown in the figure, the transfer frame 9 is slowly moved down along the drain groove 101, and the transfer frame 9 presses the open sleeve 2 downward, so that the supernatant slowly flows out through the transfer frame 9 into the other inner tube 1, thereby realizing the The separation process of the supernatant and the precipitate; in this separation process, the supernatant is discharged from the top to the bottom, which is not only easy to operate, but also difficult to turbid the supernatant, which not only improves the extraction efficiency, but also improves the DNA obtained. purity.

The invention optimizes the DNA extraction process by using a DNA high-efficiency separation tube to extract the target DNA, improves the DNA extraction efficiency, and also obtains the target DNA of higher purity, and under the premise of improving the purity of the target DNA, the molecular The accuracy of the analysis results of genetic marker technology has been further improved, so that the desired DNA type can be selected more accurately.

The above is only a preferred embodiment of the present invention; but the protection scope of the present invention is not limited to this; any person skilled in the art is within the technical scope disclosed by the present invention; according to the technical solution of the present invention equivalent replacements or changes to its improved ideas; all should be covered within the protection scope of the present invention.

Claims (10)

1. a variety screening method based on molecular genetic marker technology, is characterized in that: comprise the following steps: Step 1: Extract DNA, collect 5-10g of liver tissue from the target object, and put it into a DNA high-efficiency separation tube to extract genomic DNA; Step 2: PCR amplification, mix double-distilled water, PCR buffer, dNTP, PCR primers, TaqDNA polymerase and genomic DNA in turn, and then perform a cyclic reaction on a PCR amplifier, and purify the PCR product after the reaction; Step 3: Enzymatic cleavage, mixing sterile water, buffer, BSA, restriction endonuclease and the purified PCR product, and analyzing the enzymatic cleavage product by agarose gel electrophoresis; Step 4: Hybridization analysis, DNA fragments are transferred to a support membrane (nylon membrane or nitrocellulose membrane) by Southern blotting, and then probes labeled with radioisotopes (32P) or non-isotopes (such as digoxigenin, fluorescein) are used Hybridize with the DNA fragments on the support membrane, and finally do autoradiography to detect polymorphic bands and identify DNA differences. 2. A method for screening varieties based on molecular genetic marker technology according to claim 1, characterized in that: the DNA high-efficiency separation tube comprises an inner tube (1) and an open sleeve (2), and the open sleeve The cylinder (2) is sleeved on the outer side of the inner pipe (1), the inner wall of the open sleeve (2) is fixedly connected with a sealing sleeve (3), and the inner pipe (1) is provided with a drain groove (101). 3. A method for screening varieties based on molecular genetic marker technology according to claim 2, characterized in that: the open end of the open sleeve (2) is fixedly connected with a pair of main limit plates (4), so A secondary limiting plate (5) and a baffle plate (6) are fixedly connected to the outer end of the inner pipe (1), the secondary limiting plate (5) is fixedly connected to the lower end of the baffle plate (6), and the secondary limiting plate (5) is fixedly connected to the lower end of the baffle plate (6). The position plates (5) are slidably connected between a pair of main limit plates (4), and bolts (8) are connected between the main limit plates (4) and the auxiliary limit plates (5). 4. A kind of variety screening method based on molecular genetic marker technology according to claim 3, it is characterized in that: described main limit plate (4) and auxiliary limit plate (5) are provided with connecting holes ( 7), the bolt (8) is threadedly connected to the inside of the connecting hole (7). 5 . The method for screening varieties based on molecular genetic marker technology according to claim 3 , wherein the width of the baffle plate ( 6 ) is larger than the width of the secondary limiting plate ( 5 ). 6 . 6. A method for screening varieties based on molecular genetic marker technology according to claim 2, characterized in that: a transfer frame (9) is provided on the open sleeve (2), and the transfer frame (9) It comprises a U-shaped frame (901), a limiting groove (201) is defined on the upper end of the open sleeve (2), and the U-shaped frame (901) is matched with the limiting groove (201). 7. The method for screening varieties based on molecular genetic marker technology according to claim 6, characterized in that: a pair of side ends of the U-shaped frame (901) are fixedly connected with a main arc-shaped sheet (902) and a secondary arc-shaped sheet (903), the main arc-shaped sheet (902) is located on one side of the secondary arc-shaped sheet (903). 8. A method for screening varieties based on molecular genetic marker technology according to claim 7, wherein a pair of side ends of the U-shaped frame (901) is also fixedly connected with a sealing gasket (904), so The sealing gasket (904) is located between the main arc-shaped sheet (902) and the secondary arc-shaped sheet (903). 9 . The method for screening varieties based on molecular genetic marker technology according to claim 6 , wherein the notch width of the drain groove ( 101 ) and the notch width of the limiting groove ( 201 ) are the same. 10 . 10 . The method for screening varieties based on molecular genetic marker technology according to claim 2 , wherein: the upper side of the inner tube ( 1 ) is further provided with a sealing cover ( 10 ), and the sealing cover ( 10) It is clamped to the outer end of the open sleeve (2).

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