Genetic diversity analysis based on biochemical and molecular markers. Techniques used (isoenzymes, PCR-based analysis of DNA polymorphism , ....)
Techniques utilisée dans l'étude de la diversité génétiques des plantes
Techniques used in studies of plant genetic diversity (isoenzymes, PCR-based analysis of DNA polymorphism )
تقنيات مستعملة في دراسة التنوع الوراثي للنبات
techniques based on biochemical and molecular markers are used in many studies related to genetic
diversity studies and all biochemistry fields. Kinds of markers change from protein analysis, isoenzymes and PCR-based molecular markers, such as RAPD (generated DNA fragments).
Les analyses en biochimie et en biotechnologies necessitent le developpement de plusieurs techniques, dont l'electrophorese pour separer les proteines, isoenzymes, fragments de DNA generes par RAPD, ... See Takween.com (biochimie et applications en Biotechnologies)
The investigation of genetic diversity of many species is based on electrophoresis
technique, which allow the separation of molecules on the basis of their electric charge and their size. In many cases, electrophoresis on polyacrylamide gels is used to carry out the separation. Before this step, extracts are prepared from leaves, in the case of plants.
See more in Biotechnology techniques.
Isoenzymes. Examples of biochemical markers used in genetic diversity studies
Examples of enzyme systems revealed in date palm
Reaction mixtures for isoenzyme staining after electrophoresis.(Link:
Isoenzymes marqueurs)
CASE OF HYDROLASES
Estérases (EST):
Incubate gels 15 min in 100 ml substrate solution containing: 0,03
g Alpha-Naphthylacetate (in acetone 50%) and Tris-Hcl 0.05 M pH 7,2.
+ Rince gels 2 times with water.
Incubate 20 min in 100 ml stain solution: Fast Blue RR salt 0,14 g + Tis-Hcl pH 7.2 .,05 M
Endopeptidases (ENP):
Alpha-N-Benzoyl-DL-Arginine-Beta-Naphthylamide 80 mg (in methanol) + 2 ml Nacl 1 M + 2 ml Mgcl2 0,1 M +
5 ml acetate buffer 0,1 M pH 5,0 + Distilled Water to make 100 ml solution.Incubation 15
min and remove substrate solution.
Add 100 ml solution containing: Fast Blue K 50 mg + 2 ml Nacl 1 M + Mgcl2 0,1 M + 5 ml Acetate buffer 0,1 M pH 5.0
Incubation until visualization of bands on gels
CASE OF TRANSFERASES.
Glutamate oxaloacetate transaminase (GOT):
Incubate during 15 min gels in 100 ml substrate solution containing
250 mg Aspartic acid + 100 mg Alpha-ketoglutaric acid + 50 ml Tris-Hcl
0.5 M pH 7,2 50 ml.
Rince and add 100 ml stain solution: 200 mg Fast blue BB + 50 ml Tris-Hcl
0.5 M pH 7.2
Incubation until visualization of bands on gels
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RAPD (Random Amplified Polymorphic DNA) as a PCR-based marker.
Extraction of total
DNA
DNA extraction were achieved by CTAB (Cetyl Triméthyl Ammonium Bromide) method. DNA is recovered in the aqueous phase and precipitated with ethanol or isopropanol
DNA Quantification
DNA concentrations were estimated by spectrophotometry using the
ratio OD 260/OD 280.
One unit OD260 = 50 µg/ml
Dilution of DNA extracts.
Before their amplification by RAPD technique DNA extracts were
diluted to 10 ng/µl
DNA amplification by RAPD method
Reaction mixture:A 50 µl reaction mixture is used in the DNA amplification by RAPD. Various mix could be essayed.
Example: Reaction Mix 1:
Buffer (B) without MgCl2 (10x)...... 5 µl
MgCl2 (25 mM)........................... 5 µl
dXTP (1 mM).............................. 5 µl
Primer (2,5 µM)........................... 5 µl
Taq polymérase (0,5 unité/µl)........ 5 µl
DNA (10 ng/µl)............................ 5 µl
Distilled water .............................20 µl
Examples of primers, series A and B purshased from OPERON (sequence 5'-->3')
OPA-01.....CAGGCCCTTC
OPA-02.....TGCCGAGCTG
OPA-03.....ACTCAGCCAC
OPA-04.....AATCGGGCTG
OPA-07.....GAAACGGGTG
OPA-08.....GTGACGTAGG
OPA-13.....CAGCACCCAC
OPB-01......GTTTCGCTCC
OPB-02......TGATCCCTGG
OPB-03......CATCCCCCTG
Example of gel obtained with RAPD technique on DNA extracted from date palm leaves:
Useful links
- Techniques used in Biochemistry and Molecular biology analyzes
- Techniques d'électrophorèse
- Techniques d'isoenzymes (pdf)
- Marqueurs basés sur la PCR
- Application du marquage isoenzymatique chezlz palmier dattier. Travaux pratiques (TP)
- Téchniques (vidéos) couramment utilisées en Biotechnologies
- Cours du Master Biotechnologies et Amélioration des Plantes, Marrakech
-
Travaux pratiques Master Biotechnologies et Amélioration des
Plantes, Marrakech
-
Master course 'Plant resistance against pathogens'
- Annonces de Cours
- Annonces de Workshops
CONGRES, CONFERENCES
- Congrès
- Conférences
Faculty of Sciences, Cadi Ayyad University
Marrakech, 40000, Morocco
Email: baaziz@uca.ac.ma
Phone: 212524434649 (post 513)
Fax: 212524434669
Palmier dattier نخيل التمر
Date palm
Olivier الزيتون Olive
Pin d'Alep الصنوبر الحلبي
Aleppo pine
Atriplex القطف Atriplex
Céréales الحبوب
Cereals
Cactus الصبار Cactus