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In this article we will discuss about restriction fragment length polymorphism (RFLP).
Restriction enzymes are site-specific DNAses that cleave a DNA molecule whenever the recognition sequence, which is usually a 4-6 base palindrome, is present. Because of the enzyme’s sequence specificity, digestion of a particular DNA results in a reproducible array of fragments. RFLP, or length differences in homologous fragments between different DNA, are caused by changes in the primary sequence of the DNA.
These length differences can be the result of:
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a. A point mutation resulting in the loss or gain of restriction enzyme cut site
b. An insertion or deletion of DNA between two restriction enzyme cut sites
c. A deletion which overlaps a restriction enzyme site
d. A DNA rearrangement, where one end of the rearranged segment resides between two restriction enzyme sites. When such differences occur, they can be detected by DNA hybridization and used as molecular markers in fingerprinting or genetic studies.
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The flow diagram of RFLP analysis is shown in Fig. 9.5:
Advantages of RFLP:
a. Present everywhere,
b. Mendelian inheritance,
c. Co-dominant expression,
d. No pleiotropic effects,
e. Independent of the environment,
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f. Present at each developmental stage,
g. Long stability of cDNA probes,
h. Different loci may be identified by one probe,
i. Heterologous genes may be used as probes,
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j. Any number of probes can be produced,
k. Probes are producible for coding and silent sequences,
l. Probes show the variability of flanking sequences,
m. Several characters can be screened in the same sample.
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Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE):
The study of the banding pattern of sodium dodecyl sulphate extracts of protein in PAGE is one of the most commonly used methods to characterise plant germplasm/cultivars.
The procedure as outlined by Laemmli (1970) along with some modifications (N. K. Singh, personal communication) is as follows:
Equipment:
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1. Pestle mortar,
2. Weighing balance,
3. Water-bath,
4. Refrigerated centrifuge (10,000 – 15,000 rpm),
5. Micropipettes,
6. Tips,
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7. Spatula,
8. Forcep,s
9. Eppendorf tubes,
10. Gloves,
11. Electrophoresis system (vertical) — ATTO Corporation, Japan make or equivalent,
12. Hamelton glass micro-syringe,
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13. Transparent plastic trays.
Stock Solutions and Buffers:
1 N Tris HCl (pH 7.4 )
12.14 g Tris HCl is dissolved in 80 ml distilled water and concentrated HCl is added until pH falls to 7.4. Volume is made up to 100 ml.
2 % Sodium dodecyl sulphate (SDS)
2 g SDS is dissolved in 100 ml distilled water.
10 % Glycerol
10 ml glycerol is mixed with 90 ml water and the final volume is made up to 100 ml.
2 % Mercaptoethanol
2 ml mercaptoethanol is mixed with distilled water and the volume is made up to 100 ml.
1 mM PMSF (phenyl methyl sulphonyl fluoride)
17.4 mg PMSF is dissolved in isopropanol. Volume is made up to 100 ml and solution is stored in dark bottle.
10 % Sodium dodecyl sulphate
10 g SDS is dissolved in distilled water and the volume is made up to 100 ml.
10 % Ammonium per sulphate
1 g APS is dissolved in 10 ml of distilled water. This solution is prepared afresh before use. TEMED (N’N’N’N’ tetra methyl ethylene diamine)
As available
Extraction buffer – (10 ml)
1 N Tris HCl – 2.5 ml
2 % SDS – 2.5 ml
10 % Glycerol – 2.5 ml
1 mM PMSF – 1.5 ml
2 % Mercaptoethanol – 1.0 ml
Acrylbis
Acrylamide – 30 g
Bisacrylamide – 0.8 g
Final volume is made up to 100 ml with distilled water and stored in dark bottle.
Separating gel buffer (pH 8.8)
Tris – 12.14 g
Distilled water – 80 ml
After adjusting the pH 8.8 with HCl/NaOH, the volume is made up to 100 ml.
Stacking gel buffer (pH 6.8)
Tris – 6.1 g
Distilled water – 80 ml
After adjusting the pH 6.8 with HCl/NaOH, the volume is made up to 100 ml.
Sample buffer
Tris – 1.51 g
SDS – 2g
Glycerol – 2 ml
Mercaptoethanol – 2 ml
Bromophenol blue – 2 mg
Final volume is made up to 100 ml with distilled water.
10 X Electrode buffer (pH 8.6)
Tris – 32.5 g
Glycine – 148.0 g
SDS – 10 g
After adjusting the pH 8.6 with HCl/NaOH, the volume is made up to 1 litre. For use, 1 part of this solution is mixed with 9 parts of distilled water.
Staining solution
Solution A
Coomassie brilliant blue R-250 – 0.25 g
Distilled water – 25 ml
Solution B
Trichloroacetic acid (TCA) – 60 g
Distilled water – 25 ml
Methanol – 180 ml
Glacial acetic acid – 60 ml
Solutions A and B are mixed and the volume is made up to 1 litre. This staining solution is stored in dark bottle.
Destaining solution – (3 % NaCl)
NaCl – 5g
Distilled water – 500 ml
Gel preparation separating gel – (20 ml for 1 gel of 1 mm thickness)
Acrylbis – 8.80 ml
Separating gel buffer (pH 8.8) – 5.00 ml
Distilled water – 6.60 ml
SDS (10 %) – 440 µl
TEMED – 30 µl
APS (10 %) – 120 ml
(To be added just before pouring the gel)
Stacking gel (10 ml for 1 gel of 1 mm thickness)
Acrylbis – 3.33 ml
Stacking gel buffer – 2.50 ml
Distilled water – 4.14 ml
SDS – 100 µl
TEMED – 10 µl
APS (10%) – 60 µl
(To be added just before pouring the gel)
Protocol:
1. The seed coat is removed with the help of forceps.
2. 0.1 g seed is taken in precooled mortar and crushed with the help of pestle followed by addition of 1 ml extraction buffer.
3. The sample is transferred to the eppendorf tubes (1.5 ml).
4. The sample is incubated in water-bath for 5 min. at 100°C.
5. The sample is centrifuged at 4°C and 12,000 rpm for 35 min.
6. The supernatant containing protein is taken in eppendorf tubes and stored at 4°C/-20°C till further use.
7. Protein sample is appropriately diluted with the sample buffer (30 µl: 30 µl) and heated in boiling water bath for 5 min. at 65°C just before loading the sample.
Procedure:
The procedure described below is applicable to the Model AE 6210 slab gel cast and AE 620 dual slab chamber of ATTO Corporation, Japan. The gel dimension is 13.8 x 13 cm. The gel casting is done in horizontal position of plates.
1. The glass plates are cleaned with water.
2. The plate with notch is kept into the gasket and rubber spacer is fixed covering 3 sides (bottom, left and right sides).
3. The second plate (without notch) is mounted over the first plate and both are clamped.
4. The separating gel is poured into the space between the plates leaving about 2.5 cm space from the top.
5. Water is carefully over layered on the separating gel to have plain surface of the gel.
6. The gel is left for about 30 min. for polymerisation and the gel polymerisation can be easily known by checking the gel solution left in the beaker.
7. When the gel is fully set, a clear interface will be visible between the water layer and the gel.
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8. The excess of water is removed from the top of the gel.
9. The stacking solution is poured between the glass plates and a comb with adequate number of teeth is inserted to make the wells.
10. The gel is left for about one hour for complete polymerisation.
11. After the gel is completely set, the comb is removed carefully and the gel is transferred to the electrophoretic tank.
12. Electrode buffer is filled into the upper and lower chambers.
13. The protein sample (5 µl) is loaded in the wells of the gel with a micropipette.
14. Protein molecular weight marker is used along with the samples.
15. The run is performed at a constant voltage of 100 volts.
16. The run is stopped when the dye has reached approximately 0.5 cm from the bottom of the separating gel.
17. It takes about 4-4.30 hours for complete run.
18. The gel is removed with the help of spatula and the gel is marked by cutting the corner of the bottom side towards the last sample to have identity of samples.
19. The gel is transferred to the staining solution for overnight.
20. The staining solution is replaced the next day with destaining solution.
21. The gel is shaken and the destaining solution is changed 3-4 times till the blue background of the gel disappears.
22. The gel is visually examined and the diagram is drawn showing position and intensity of bands. It should be photographed and documented in a gel documentation system if available. The gel is rinsed in distilled water to remove excess of salts, if any and kept in plastic bags which are sealed. This gel is stored in refrigerator for future reference, if needed.