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A procedure for the determination of fragmentation of DNA in animal spermCM Patents

[0001] The present invention describes a method for the determination of DNA fragmentation in animal sperm cells. Specially it is related to a method to evaluate the integrity of the chromatin/DNA of the sperm cells by means of the sample treatment with a denaturing DNA solution followed, optionally, of a staining; a later treatment with a lysing solution which does not contain protein denaturing detergent, followed optionally by a staining; and an evaluation of the integrity of the chromatin/DNA. The present invention is furthermore related to a kit to evaluate the quality of the animal sperm cells which includes a denaturing DNA solution and a lysing solution which does not contain protein denaturing detergent.

Datos clave

CategoríaInvención

Índice de la ficha

Updated at
05/11/2026
Numero publicacion
EP.1710320.A1
Fecha publicacion
11/10/2006
Anio publicacion
2006

En detalle

Reivindicaciones

1. Method to evaluate the integrity of chromatin/DNA and animal sperm comprising: a) a treatment step of the sample containing the sperm, with a solution of DNA denaturing solution, b) a single treatment step with a lysis solution to extract the nuclear proteins, c) an evaluation stage of the integrity of the chromatin/DNA of the sperm characterised because the lysis solution does not contain protein denaturing detergents and essentially does not destroy the tails of the sperm. 2. Method according to claim 1 characterised in that stage a) precedes that of b), or it only proceeds to b) and c). 3. Method according to claim 1 or 2, characterised in that the lysis solution comprises of a non-ionic non protein denaturing detergent. 4. Method according to claims 1-3 characterised in that the non ionic detergent is selected from the group toctylphenoxypolyethoxyethanol (Triton X- 100), N , N-bis(3-D-Gluconamidopropyl) cholamide (bigCHAP), Brij(r) 35 P, N- decanoyl-N-methylglucamine, digitonin, dodecanoyl-N-methylglucamide, heptanoyl-N- methylglucamide, branched octylphenoxy poly (ethyleneoxy) ethanol (Igepal CA-630), N-Nonanoyl-N-methylglucamine, Nonidet P 40, N- Octanoyl-N-methylglucamine, Span 20 solution, Polysorbate 20 (Tween 20) and their mixtures, preferably Triton X-100. 5. Method according to claims 1-4 characterised in that the lysis solution comprises sodium chloride between 1 and 3M, dithiothreitol (DTT) between 0.001 and 2M, 2-amino-2 (hydroxymethyl)-1,3-propanediol (Tris) between 0.001 M and 2 M and Triton X-100 between 0.1 % and 3%. |EPO DP num="38"| 6. Method according to claims 1-5 characterised in that the lysis solution comprises 2.5M sodium chloride, around 0.2M DTT, around 0.2M Tris, around 1 % Triton X-100 and a pH of around 7.5. 7. Method according to claims 1-6 characterised in that the DNA denaturing solution is acid. 8. Method according to claim 7 characterised in that the DNA denaturing solution comprises an acid selected from the hydrochloric, acetic, nitric acid group or mixtures of these. 9. Method according to claim 8 characterised in that the DNA denaturing solution comprises hydrochloric acid. 10. Method according to claims 1-9 characterised in that after steps a) and b) there is a sample staining step. 11. Method according to claim 10 characterised in that the staining is made with a Wright type solution. 12. Method according to claims 1-11 characterised in that the sample containing the sperm is included in a medium similar to a suspension, preferably in a microgel. 13. Method according to claim 12 characterised in that the sample containing the sperm is included in an agarose microgel. 14. Kit for the evaluation of the quality of the sperm of animals which comprises: a) a DNA denaturing solution, b) a lysis solution to extract nuclear proteins, characterised in that the lysis solution does not contain a protein denaturing detergent and essentially does not destroy the tails of the sperm. |EPO DP num="39"| 15. Kit according to claim 14 characterised in that the lysis solution comprises sodium chloride between 1M and 3M , dithiothreitol (DTT) between 0.001M and 2 M, 2-amino-2 (hydroxymethyl)-1,3 propanediol (Tris) between 0.001M and 2 M and Triton X-100 between 0.1% and 3%.

Etiquetas

Inventores
Gosalvez Berenguer JaimeGosalvez Berenguer, JaimeFernandez Garcia, Jose LuisFernandez Garcia Jose LuisGoyanes Villaescusa VicenteGoyanes Villaescusa, VicenteJose Luis Fernandez GarciaGarcia Jose Luis FernandezVillaescusa Vicente GoyanesVicente Goyanes VillaescusaBerenguer Jaime GosalveJaime Gosalve BerenguerBerenguer Jaime GosalvezJaime Gosalvez BerenguerGosalves Berenguer, JaimeGosalves Berenguer JaimeGosalvez Berenguer Jaime,Fernandez Garcia Jose Luis,Goyanes Villaescusa VicenteVicente Gosalvez Berenguer JaiGOSALVEZ BERENGUER JaimeFERNANDEZ GARCIA Jose LuisGOYANES VILLAESCUSA VicenteGosalvez BerenguerGosalvez, Berenguer JaimeFernandez, Garcia José LuisFernandez Garcia José LuisGoyanes, Villaescusa VicenteГОСАЛЬВЕС БЕРЕНГЕР Хайме(ES)Gosal Ves Berenger KhajmeFernandes Garsija Khose LuisФЕРНАНДЕС ГАРСИЯ Хосе Луис(ES)ГОЯНЕС ВИЛЬЯЭСКУСА Висенте(ES)Gojanes Vil Jaehskusa VisenteGosalvez Berenguer Jaime Univ Autonoma Madrid
Solicitantes
Universidad Autónoma de MadridУниверсидад Аутонома Де Мадрид
Clasificacion ipc
G01N 33/ 50 A IC12Q 1/ 68 A IC12N 15/ 10 A IC12Q 1/ 6806 A IC12Q 1/ 02 A IG01N 33/ 48 A I
Clasificacion cpc
2G045/AA242G045/BA142G045/BB022G045/BB232G045/BB252G045/BB292G045/BB362G045/BB502G045/BB512G045/DA122G045/DA132G045/FA162G045/FA182G045/FB022G045/FB122G045/FB162G045/GC014B063/QA054B063/QA194B063/QQ084B063/QQ424B063/QR084B063/QR524B063/QS114B063/QS364B063/QX01C12Q1/02C12Q1/68&AC12Q1/68&ZG01N33/48&AG01N33/48&PG01N33/50&J435/6.1435/6.19536/22.1
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