77 results about "Sperm dna" patented technology

Devices, compositions, and methods for handling, separating, packaging, and utilization of spermatozoa (1) that can be derived from previously frozen sperm samples collected from a male mammal. Specifically, techniques to uniformly stain (2) spermatozoal DNA even when derived from previously frozen sperm and separation techniques to separate and isolate spermatozoa even when derived from previously frozen sperm samples into X-chromosome bearing and Y-chromosome bearing populations having high purity.

This invention is a method of separating mammal XY sperm. Fluorescence colorant Hoechst33342 is used to color sperm DNA and then they are separated by flow cytometry according to theory of DNA content of X sperm is more than Y sperm of mammal. Preservation method, sperm diluent, sheath liquor and acceptance liquor used for separating sperm before sperm separating is improved to confirm improve the separation accuracy rate and activity after separation at the same time of ensuring separating XY sperm efficiency. It is fit for big scale commercialization separating generation of XY sperm of scalper, buffalo, goat and other mammal. It can be bonded to artificial insemination and external insemination and other animal breeding new techniques, it greatly improved the efficiency of animal husbandry production.

In various aspects, the present invention provides novel and effective methods and kits for the isolation of sperm and sperm DNA from samples having at least one other cell type, or having the DNA of one other cell type. More specifically, a process for isolating sperm DNA from a mixture of sperm and non-sperm cells by filtration is provided. DNA from non-sperm cells is solubilized by selective lysis, and the intact sperm are retained on a filter, washed, and then treated in situ with a reducing agent to solubilize the sperm DNA. Significantly, centrifugation steps are not required, the process can be easily automated, and can be performed on many samples in parallel. The novel methods and kits are based on filtering selectively solubilized samples through filters that retain sperm by virtue of having uniform pore diameters that: are smaller than sperm, large enough to allow passage of cell debris and solubilized DNA; and are stable to pressure. The inventive methods and kits have broad utility, particularly in the forensic art.

The invention provides a human sperm DNA fragment detection method comprising the following steps: (1) diluting liquefied sperm with a buffer solution of 4 DEG C; (2) adding the diluted sperm into a sample tube of a flow cytometry; (3) adding an acid treatment buffer solution; (4) after 30 seconds, adding an AO staining solution; (5) setting a calibration and flow chamber of the flow cytometry, equilibrating a sample line by an AO equilibration buffer solution; (6) continuously determining each sample at least twice, recording at least 5000 cells and carrying out statistical analysis; and (7) after determining, respectively thoroughly cleaning the cell fragments and fluorescent dye remaining in the sample line of the flow cytometry by bleacher, sample tube detergent and degerming double distilled water. A human sperm DNA fragment detection kit comprises 50mL of staining buffer solution, 300mu L of AO stock solution, 20mL of acid treatment solution, 100mL of buffer solution, 100mL of sheath fluid, 100mL of AO remover, 100mL of sample tube detergent and 100mL of double distilled water (ddH2O). The invention realizes the purpose of clinically detecting the sperm DNA fragments simply, conveniently, quickly and accurately.

The invention provides a sperm DNA fragmentation detection kit. The kit comprises an enveloped glass slide, a fusible gel, an A solution, a B solution, a Wright stain, a Wright buffer solution and a SCD (sperm chromatin diffusion) storage solution. The invention further provides a method for detecting sperm DNA fragmentation by using above kit. The reagent structure is optimized according to the detection method so that the reagent is stable and reliable; the operation of detecting by using the reagent is simple and free from special detection instruments, the clinical conventional application and popularization are facilitated; by adopting the unique SCD storage solution, the sperm sample can be effectively stored, the DNA fragmentation rate cannot be changed after being cryopreserved at -20 DEG.C within two weeks, the required sample can be easily stored through the SCD storage solution, the detection can be performed without the limitation of time and number, and large number of manpower and material resources are saved.

The invention provides a method for extracting the genomic DNA of the frozen semen of bull, which comprises the steps of washing of the semen of the bull, lysis and digestion of the semen and purification of the genomic DNA. In the method, the digestion yield of the DNA of the semen of the bull is increased by improving the components and mixing ratio of digestive lysis solution and determining proper digestion time; meanwhile, instead of the conventional method for purification by organic solvent extraction, a simpler high-salt method is used to purify DNA, and the high-salt method has the advantages of using easily prepared reagent, effectively removing impurities, obtaining high-yield and high-quality genomic DNA of frozen semen, along with low cost and suitability for high-efficiency and batch preparation of genomic DNA of frozen semen.

The invention discloses a method for detecting sperm DNA fragmentations by a sperm chromatin dispersion test, which comprises the following steps that: 1) a sperm lysis solution is accurately prepared; 2) the sperm lysis solution is placed at a temperature of 4 DEG C and is preserved for 2 weeks, is taken out 1.5 hours earlier before the use, and is placed at a room temperature of 22 DEG C; 3) semen is diluted to 5-10x10<6> / ml; 4) 50 microlitres of diluted semen is taken and placed in a refrigerator for 5 minutes, and the temperature in the refrigerator is 4 DEG C; 5) a microscopic glass is removed after the diluted semen is taken out from the refrigerator, and a glass slide is horizontally immersed into hydrochloric acid for degradation for 7 minutes, and then is taken out; 6) the glass slide taken away in the step 5) is horizontally immersed into the sperm lysis solution in the step 1) for 20 minutes; 7). The glass slide in the step 6) is washed by a buffer liquid for 5 minutes; 8) alcohol is used to respectively dehydrate for 2 minutes; and 9) a Wright method or a Giemsa-Wright method or a sky blue method is used to dye sperms, and the sperm DNA fragmentations are observed and analyzed under an optical microscope. The method realizes the aims of simplicity, quickness, accuracy, low cost, high resolution of sperm DNA detection.

The present application discloses a method for pre-treating a sample of a DNA / RNA adsorbing matrix (e.g. soil) suitable for extracting cell-derived (deoxy) ribonucleic acid therefrom, comprising the steps of: isolating a sample of a DNA / RNA adsorbing matrix, said sample comprising DNA / RNA-containing cells; and bringing the sample into contact with DNA of natural origin in a fragmented form (e.g. salmon sperm DNA) so as to essentially block said DNA / RNA adsorbing binding sites of said adsorbing matrix, said fragmented DNA being “unrelated” to the target DNA / RNA of said DNA / RNA-containing cells. The fragmented DNA of natural origin can be depurinated and / or freeze-dried. The application further discloses a method for extracting DNA / RNA from a sample and a kit useful for pre-treating samples.

The invention relates to a method for detecting sperm DNA fragments, comprising the following steps: 1) adjusting sperm concentration; 2) completely melting low melting point gel, and balancing for standby use; 3) mixing the sperm and the gel following proper proportion; 4) absorbing the obtained suspension to solidoid enveloped with high-melting point gel, and adding a cover plate to the suspension to solidify the suspension until the temperature thereof is 2-8 DEG C after standing; 5) removing the cover plate and soaking the enveloped solidoid horizontally into denatured liquid for reaction under the condition of the temperature being lower than the temperature of the low melting point gel; 6) transferring the enveloped solidoid to solution for reaction under the condition of the temperature being lower than the temperature of the low melting point gel; 7) washing the enveloped solidoid; 8) carrying out continuous dehydration following ethanol concentration sequence from low to high, and drying in the air; 9) dyeing; and 10) counting incidence rate of the DNA fragments under a microscope. The device for detecting sperm DNA fragments comprises an enveloped solidoid, low melting point gel aqueous solution, denatured liquid and washing liquid. The outstanding technological effects are: the result is easy to be judged, the operation is easy and clinical promotion and application are easy.

The present invention relates to a detection method of gene promoter methylation. Said method includes the following steps: firstly, using freshly-prepared modification reagent to modify extracted DNA, after the modified DNA is passed through column, making purification and desulfurization reaction, then using absolute ethyl alcohol and salmon sperm DNA to enrich micro modified DNA, further selecting and using specific PCR GC Buffer to make multiple methylation specific PCR amplification, then adopting electrophoresis technique to analyze result.

The invention discloses a preparation method and application of an H1, H2 or J type astaxanthin aggresome water dispersion system. Natural biological macro chitosan and special molecular structures offish sperm DNA are adopted by astaxanthin aggresome water dispersion systems with three colors to induce the formation and stabilizing of an astaxanthin polymer through physical interaction among macro molecules combining with a solvent and a salt ion effect. Ethanol with low toxicity is taken as the astaxanthin good solvent, the organic solvent can be completely removed in the later period of the preparation process and can be further enriched and recycled to facilitate clean production and the reduction of cost. The H1, H2 or J type astaxanthin aggresome water dispersion system can be controlled by regulating technological parameters to allow the developing range of astaxanthin waterborne product to be expanded to yellow, orange and pink, and a stable aggregation state and developing effect of the astaxanthin are maintained in concentration, dehydration and reconstitution process. The preparation technology of the three kinds of the astaxanthin aggresome water dispersion systems issimple, the reaction conditions are mild, the production cost is low, the technology is green and has no pollution, and therefore industrial popularization is convenient.

The invention discloses a sperm DNA fragment detection kit and a detecting method thereof. The kit comprises mixed liquid of liquid I, hydrochloric acid, liquid II, sodium chloride, ethylenediamine tetraacetic acid, trihydroxymethyl aminomethane, sarcosyl, polyethylene glycol octylphenol ether and 3-sulfydryl-1, 2-propylene glycol, and further comprises a glass slide covered with agarose, low-gelling temperature agarose, Wright's stain, Wright's buffer solutions and the like. The detecting result reliability, sperm staining quality and the like of the kit are greatly improved. An image of the detecting result of the kit is clear, formed halos are clear, a boundary of the halos and a head is obvious, and the sperm DNA injury degree can be judged conveniently. According to the kit and the detecting method of the kit, standardization of a laboratory is facilitated, analysis costs are low, no special instrument or reagents are needed, the application range is wide, the detecting needs of small and medium laboratories are met, and the kit and the method of the kit can be suitable for detecting an DNA fragment of a single sperm. The kit can be compatible with a later automatic instrument, and is used for detecting the large laboratories, and detecting efficiency is improved.

The invention relates to a raman spectrum-based fast measurement method for a motile sperm deoxyribonucleic acid (DNA). The invention adopts the following technical scheme: collecting a semen sample by a test tube; naturally liquefying semen in a room-temperature environment; absorbing the liquefied semen and adding to a centrifugal tube containing density gradient centrifugate; absorbing upper seminal plasma after centrifuging, discarding, then adding a PBS buffer solution and centrifugally washing again; absorbing supernatant, discarding, adding the PBS buffer solution to prepare a sperm suspension, and incubating; attaching a metal bottom plate to a culture dish; adding the PBS buffer solution, and absorbing the incubated sperm suspension, soaking in the culture dish, wherein the sperm swims outside and contacts the metal bottom plate; setting a spectrum response range, integrating time and the excitation light wavelength, so as to obtain raman spectrum data from the head part of the sperm. The motile sperm swimming out from the pointed-end part of a sucker can be attached to the metal bottom plate by operation of a thin tube pipette, and the damage condition of the sperm DNA is evaluated by measuring the sperm of which the head is attached to the metal bottom plate.

A method for distinguishing individuals in mixed seminal stain by single sperm capture and mitochondrial DNA typing mainly solves the technical problems in the prior art that DNA content can not meet the requirements of routine mixed seminal stain test and that complete individual genetic information can not be provided. The method is realized by the steps of single sperm capture, DNA extraction from the single sperms, nested amplification on mt DNA HV I zone, DNA sequence analysis of the products from two amplifications and sperm concentration and autosome STR detection. According to the invention, single sperm mitochondrial DNA with personal characteristics is employed as a detection index, the mixed semen from different individuals is distinguished according to individual semen, and then nuclear DNA detection is carried out, thereby successfully solving the problem of recognizing individuals in mixed sample with components from different individuals. The characteristic of abundant mitochondrial DNA content of single sperm is utilized to meet the requirements of mitochondrial DNA detection by PCR technology; and a plurality of sperms with mitochondrial DNA of the same type are collected for realizing nuclear DNA detection, in order to achieve the purpose of individual identification.

The present invention discloses food containing haematococcus pluvialis and trehalose. The food contains the haematococcus pluvialis and trehalose, wherein a mass ratio of the haematococcus pluvialisto the trehalose is (100-800):(500-1,800). The food can increase spermatogenesis, promote sperm proliferation and maturity, energize sperms, increase sperm vitality, protect integrity of sperm cell membranes, increase stability of sperm DNA, comprehensively improve quality of semen and receive satisfactory results.

The invention discloses a method for identifying sperm fragments in a DNA image. The method comprises the following steps: collecting a sperm image of a sperm sample, carrying out image preprocessingon the sperm image to screen out a plurality of candidate sperms, extracting the contour of the sperm head of each candidate sperm, calculating the nuclear contour and halo contour of the sperm head of each candidate sperm, and calculating the halo contour of the sperm head of each candidate sperm; fitting the radius of the outline of the sperm head of each candidate sperm and classifying each candidate sperm; and classifying DNA fragments of normal sperms and existing sperms from the sperm image. Therefore, a doctor can further analyze the causes of infertility, spontaneous abortion and embryo infertility from the DNA fragments with sperms, and can evaluate and diagnose the outcome of pregnancy and the selection of an auxiliary fertility mode in advance.

The invention discloses a sperm nuclear protein lysate; every 100ml of distilled water contains 4.844g of trishydroxymethylaminomethane, 11.7g of sodium chloride, 1.86g of dithiothreitol, 8 to 15g of Gleditsia Sinensis Lam as well as 12.32g of ethylenediaminetetraacetic disodiumsalt; the pH value is neutral. The Gleditsia Sinensis Lam extractive contained in the lysate of the invention does not lead the protein to be modified and does not damage the tail part of the sperm. The lysate of the invention can simply, fast and accurately detecte the DNA of the sperm with low cost and high resolution.

The invention relates to a preparation method for artificially synthesizing a necessary DNA raw material, and belongs to the technical field of biosynthesis and biocatalysis of important chemicals. Brand-new enzyme hydrolysis and enzyme catalysis processes are adopted, mackerel sperm DNA is selected and used as a raw material, the mackerel sperm DNA is hydrolyzed by phosphodiesterase to generate adNMP mixture, ion-exchange chromatographic separation and purification are performed, a dNTP mixture is synthesized by whole-cell catalysis of active yeasts, then four high-purity monomeric compoundsare prepared by preparative chromatographic separation and purification, and compounding is performed to form an equimolar dNTP biochemical reagent. The process is environment-friendly in reaction, efficient, low in cost, suitable for industrial production, and extremely competitive, and the product purity reaches 99.9% or above.

The invention discloses an evaluation method for the survival rate and the quality of prawn sperms. The evaluation method is applicable to evaluating the survival rate of prawns treated by a chemical method and the damaging degree of the sperms of the prawns; a mixed biological dye of eosin and nigrosine is used for clearly distinguishing dead and live sperms under an optical microscope and the survival rate of the sperms is counted; a single sperm dyed by GelRed is fixed by using single cell gel and sperm DNA (Deoxyribonucleic Acid) is dispersed by electrophoresis; a migration track of the DNA of the damaged sperm can be observed under a luminescence microscope and the comet rate and the damaging coefficient are counted by CASP (Computer Automated Stowage Planning) analysis software so as to further evaluate the damaging degree and the quality of the prawn sperms. According to the evaluation method, a suitable mixed biological dyeing solution is adopted and cells are clearly distinguished after being dyed; a background color has small interferences on observation and a dyeing result is easy and rapid to read; the result is accurate and specific; a single cell gel electrophoresis method can further identify the damaging condition of the sperms and can effectively and accurately obtain evaluation information of the survival rate and the quality of the prawn sperms.

The invention relates to a method for evaluating male infertility by combining sperm DNA fragmentation index, reactive oxygen species and 8-hydroxydeoxyguanosine. The method comprises the steps of: determining levels of sperm DNA fragmentation index (DFI), reactive oxygen species (ROS) and 8-hydroxydeoxyguanosine (8-PHdG) from semen samples; correlating levels of the sperm DNA fragmentation index,reactive oxygen species and 8-hydroxydeoxyguanosine with sperm function quality. With sperm DFI=19.26%, ROS=1.91ng / ml and 8-OHdG=50.76ng / l as judgment critical values, the sperm is judged to be infertile when the sperm DFI, ROS and 8-OHdG levels are higher than the critical values.

The invention provides a multifunctional sperm quality analysis system. The multifunctional sperm quality analysis system comprises sperm routine computer-aided sperm analysis (CASA) and image analysis, the CASA is used for analyzing a sample video recorded through a CCD camera or video inputted into the local of an analysis host to calculate the quantity, the concentration, the vitality and the motion trail of sperms of the sample; the image analysis is used for analyzing sperm functions and comprises sperm DNA fragment detection, sperm nucleoprotein detection and sperm morphological detection, and pictures taken through the CCD camera or pictures inputted to the local of the analysis host are analyzed. The problems that existing seminal fluid analysis operation is tedious, human factorsare uncontrollable, and the inspection cost is high are solved.

The invention discloses a diluent formula for improving the frozen quality of yak frozen semen. Each 100 milliliters of frozen semen diluent is prepared from 0.9 to 1.2 grams of sodium citrate, 3 to 5grams of saccharose, 6 to 8 milligrams of low density lipoprotein, 4 to 7 milliliters of glycerinum, 6 to 8 milligrams of resveratrol, 5 to 8 milligrams of melatonin, 35 milligrams of spectinomycin and 80 thousand units of gentamicin. According to the diluent formula disclosed by the invention, the low density lipoprotein, the resveratrol and the melatonin are jointly added into a semen diluent for the first time, and a diluent preparation method is corrected. Experimental data show that the frozen semen diluent not only can improve sperm motility and acrosomal intactness rate of frozen-thawed semen, but also improves activity of catalase (CAT), glutathione peroxidase (GR) and glutathione reductase (GSH) in seminal plasma which are used for protecting sperm fertilizing ability; furthermore, sperm DNA integrity is improved, and the fact that the semen formula can improve the frozen quality of semen is ensured.

The invention provides a protective agent, a preservation method and application of a low-concentration DNA reference material. The protective agent comprises salmon sperm DNA. A sequence of a TP53 gene with the length of 300 bp is selected as a target fragment to develop a DNA reference material with a low-concentration level, the salmon sperm DNA with the concentration of 20 [mu]g / mL to 100 [mu]g / mL is taken as a protective agent for protecting, a the DNA reference material is stored in a low-absorption centrifuge pipe and placed in a freezing condition of -15 DEG C to -20 DEG C, and the concentration stability of the DNA reference material is maintained within 6 months; the DNA reference material has good uniformity and stability, the accurate reliability of a biological frontier detection technology are ensured, the detection ability of clinical detection and early diagnosis of cancer is improved, and the reliable measurement support is provided for precision medical plans.

A method is provided herein, wherein the method of capturing a sperm deoxyribo nucleic acid (DNA) in a sample, comprises contacting a lysis solution to the sample comprising at least a sperm cell or a sperm cell lysate to lyse the sperm cell. The sperm cell or sperm cell lysate comprises a protamine-DNA complex. The method further comprises applying at least a protamine-specific antibody to the lysed sperm cell, wherein the protamine-specific antibody binds to the protamine-DNA complex of the lysed sperm cell to form an antibody-protamine-DNA complex. The antibody binding is followed by capturing the antibody-protamine-DNA complex; and isolating and detecting the sperm DNA from the captured antibody-protamine-DNA complex.

The invention relates to the field of fish sperm preservation solutions, in particular to a pseudobagrus sperm preservation solution for solving the problem of pseudobagrus large scale reproduction and a preparation method of the pseudobagrus sperm preservation solution. The sperm preservation solution is prepared from sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium bicarbonate, glucose and penicillin and is prepared by weighing the raw materials, adding the raw materials in a plastic bottle or a glass bottle, and preparing into a solution with distilled water. The prepared sperm preservation solution is subjected to sperm chromatin detection with a flow cytometer, spermatids are circled on an FSC / SSC graph, and a door is set on an FL1(FITC) / FL3(ECD) scatter diagram for analysis; and a DNA fragment index at is calculated through a formula to evaluate sperm DNA quality. The sperm preservation solution provided by the invention can be preserved for 10 days in a refrigeration tank at the temperature of 4 DEG C, 98 percent or more of sperms keep exuberant vigor and can still be taken as sperms for semination, and the fertilization rate is 95 percent orabove.

The invention provides a method and a kit for detecting sperm DNA fragment rate. According to the method and the kit provided by the invention, RNA in a sperm sample is digested by RNA enzyme based onflow cytometry, so that mixed signals brought by sperm RNA are eliminated, the detection result is more accurate, stable and reliable, the repeatability is good, and the clinical popularization is easy.

An isolated nucleic acid, a recombinant protein encoded thereby and uses thereof. The isolated nucleic acid including (a) a polynucleotide at least 60% identical to SEQ ID NOs:1, 3, 5 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty-3); (b) a polynucleotide encoding a polypeptide being at least 60% homologous with SEQ ID NOs:2, 4, 6 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty-50, gap extension penalty-3); or (c) a polynucleotide hybridizable with SEQ ID NOs:1, 3, 5 or portions thereof at 68° C. in 6xSSC, 1% SDS, 5x Denharts, 10% dextran sulfate, 100 mug / ml salmon sperm DNA, and <32>p labeled probe and wash at 68° C. with 3xSSC and 0.1% SDS.

The invention relates to the technical field of biology, and particularly relates to a sperm DNA extraction method. The method comprises the following steps of: 1) taking a semen sample, cleaning thesemen sample, and performinglysis on the semen sample with a lysis buffer to obtain a lysate; 2) uniformly mixing the lysate with an alcohol solvent, and collecting a precipitate; and 3) washing the precipitate obtained in the step 2) with a sodium citrate solution, and collecting a precipitate, wherein the lysis buffer is prepared from 4.03-4.45M of guanidine thiocyanate, 0.095-0.105M of sodium chloride, 0.95-1.05% of sodium dodecyl sarcosinate, 0.1425-0.1575M of dithiothreitol and 0.19-0.21mg / mL of protease K, and is prepared by using sterile water. The sperm DNA extraction method provided by the invention is short in extraction time, large in total amount and high in quality, and can meet the requirements of various subsequent molecular genetic experiments.

This invention comprises peptides, methods and a kit for identifying DNA fragmentation in non-viable sperm. Specific peptide sequences are claimed that bind to ssDNA and fragmented DNA.