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, ...

TECHNIQUES 1. EDUCATION. See TAKWEEN.COM .

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.

Preparation of extracts used in electrophoresis

TECHNIQUES 2. EXAMPLES OF BIOCHEMICAL & MOLECULAR MARKERS USED IN GENETIC DIVERSITY STUDIES

ISOENZYMES. EXAMPLES OF ENZYME SYSTEMS REVEALED IN DATE PALM

REACTION MIXTURES FOR ISOENZYME STAINING AFTER POLYACYLAMIDE GEL ELECTROPHORESIS.

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

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

RAPD (Random Amplified Polymorphic DNA).

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 OD260/OD280. 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

RAPD markers in date palm

PCR program:
Predenaturation at 94°C for 4 min
Denaturation at 94°C for 1min
Annealing at 35°C for 1 min
Elongation at 72°C for 1 min 30 seconds
Post cycle at 72°C for 10 min
Storage at +4°C

Electrophoresis on polyacrylamide gel
Polyacrylamide gels (7%) prepared in TBE 10 x were used to separate DNA fragments amplified by RAPD. After electrophoresis at 150 volts during 4 hours, gels were stained by silver nitrate (Bassam et al., 1993). Hind III digest of lambda phage DNA is used as molecular weight markers.