SilkSatDB-A Silkworm Microsatellite Database


*SilkSatDb: The First Comprehensive Database on Insect Microsatellites*

Analysis of Inter Simple Sequence Repeats (ISSR)

Analysis of Inter Simple Sequence Repeats (ISSR)

PRINCIPLE

The simple sequence repeats or microsatellites especially (TAA)_n and (AT)_n repeats are ubiquitous in the silkworm genome. The changes in the number of repeats in a microsatellite array, which is considered to be the result of slippage of the DNA polymerase which occurs during DNA replication, provide an unlimited source of polymorphism. However, studying SSR is quite labor intensive since complete sequence information flanking the repeats is necessary to design primers for PCR amplification. Here, we demonstrate an alternative, efficient method that does not require any prior sequence information. In this method microsatellite sequence anchored either at 5' or 3' ends with a stretch of degenerate nucleotides were used for inter-repeat region amplification.

ADVANTAGES

This method requires small quantity of DNA (5-20 ng/reaction depending up on the type of electrophoretic assay) and it is PCR based.
This method provides dominant, reproducible and large number of markers.
The whole analysis is automated to enable the researchers to carry out large-scale genetic mapping and population studies.
This method overcomes the limitation of flanking sequence characterization as required by SSRs.

LIMITATIONS

It is a dominant marker and hence not as informative as SSRs.

Click on the image to enlarge

ISSR-PCR. A single primer targeting a (CA)_n repeat, anchored either at the 3' (green arrows) or at the 5' end (yellow arrows) of the repeat, is used to amplify genomic sequence flanked by two inversely oriented (CA)_n repeat sequence.

Inter Simple Sequence Repeat region can be amplified using different protocols as per the lab’s requirement. In the past years we have tested many different protocols and standardised them for our population studies, polymorphism estimations, strain/variety identification, Mapping etc. Here, we list the best of our methods, which are tried and tested, in our lab for your use.

Protocols List:

Protocol of ISSR PCR for agarose gel electrophoresis, is a fast and efficient technique for standardising primers and quick polymorphism testing including mapping. This method is efficient in differentiating products of 2% difference. The only drawback is the possibility of missing faint amplification products, as the sensitivity of detection is low.
Protocol of ISSR PCR for PAGE and silver staining, is an efficient and reliable method with a better resolving power than agarose gel. It is safer as it does not require radioactivity but is more labour intensive than agarose gels.
Protocol of ISSR PCR using radiolabel, is the most sensitive technique among these methods. It is labour intensive and involves the risk of radioactivity handling.
Protocol for fluorescent ISSR (FISSR) PCR, combines both speed and sensitivity but is more expensive than other methods. This method is automated with the use of fluorescent dNTPs or fluorescent labelled primers and thus requires automated sequencing system. It is specially suitable for large scale genetic analyses.

I.Protocol of ISSR PCR for agarose gel electrophoresis

1. Equipment and reagents

a) Thermal cycler

b) Poly Acrylamide Gel Electrophoresis system

c) The reagents including Taq DNA polymerase, 10 x PCR buffer (500 mM KCl, 100 mM Tris-HCl, 0.01% gelatin and 1% Triton X-100), 10 x dNTPs stock (1 mM) and 10 ng DNA samples.

d) 1 x TBE (90 mM Tris borate, pH 8.3, 2 mM EDTA)

e) Silver staining reagents like, fixative-stop solution (10% ethanol), silver nitrate solution (0.2% silver nitrate in water), developer solution (1.5% sodium hydroxide and 3ml/l formaldehyde).

2. ISSR PCR reaction mix

Template DNA	1 ml (10 ng)
10 x PCR buffer	1 ml (1x)
25mM MgCl₂	0.6 ml (1.5mM)
1mM dNTPs	1 ml (100 mM)
4 mM ISSR primer	1 ml (0.4 mM)
Taq Polymerase	0.1 ml (0.5 U)
Sterile water	5.3 ml
Total volume	10 ml

3. Thermal Cycling Conditions

Initial denaturation	94^oC for 2 min.

Denaturation	94^oC for 30 sec.	35 cycles
Annealing	50^oC* for 30 sec.
Extension	72^oC for 1 min.

Final extension	72^oC for 10 min.

4. Agarose gel electrophoresis conditions

Aliquots of amplified DNA from individual PCR reactions should be loaded on a denaturing 2.5% (3 parts of Metaphor agarose: 1 part of agarose) gel in 1x TBE. Electrophoretic separations should be performed in 1x TBE in a horizontal gel tank.

II. Protocol of ISSR PCR for PAGE and silver staining

1. Equipment and reagents

a) Thermal cycler

b) Poly Acrylamide Gel Electrophoresis system

c) The reagents including Taq DNA polymerase, 10 x PCR buffer (500 mM KCl, 100 mM Tris-HCl, 0.01% gelatin and 1% Triton X-100), 10 x dNTPs stock (1 mM) and 10 ng DNA samples.

e) Silver staining reagents like, fixative-stop solution (10% ethanol), silver nitrate solution (0.2% silver nitrate in water), developer solution (1.5% sodium hydroxide and 3ml/l formaldehyde).

2. ISSR PCR reaction mix

Template DNA	1 ml (10 ng)
10 x PCR buffer	1 ml (1x)
25mM MgCl₂	0.6 ml (1.5mM)
1mM dNTPs	1 ml (100 mM)
4 mM ISSR primer	1 ml (0.4 mM)
Taq Polymerase	0.1 ml (0.5 U)
Sterile water	5.3 ml
Total volume	10 ml

3. Thermal Cycling Conditions

Initial denaturation	94^oC for 2 min.

Denaturation	94^oC for 30 sec.	35 cycles
Annealing	50^oC* for 30 sec.
Extension	72^oC for 1 min.

Final extension	72^oC for 10 min.

4. PAGE electrophoresis conditions

Aliquots of amplified DNA from individual PCR reactions should be loaded on a denaturing (7M Urea) 6% Poly Acrylamide sequencing Gel in 1x TBE. After the bromophenol blue dye runs out, the gel is silver stained for band detection.

5. Silver staining

After electrophoresis, gels should be fixed in fixative-stop solution for 30 min. Fixed gel must be rinsed 3 times with water for 2 min. each. Later gel must be impregnated with silver nitrate solution for 10 min. and rinsed with distilled water for 5-20 sec. Gel can be developed with cold developer solution for 4 min. Developing reaction should be stopped with fixative-stop solution for at least 1 min. and washed extensively with water. Dry stained gels at room temperature and store in photographic albums.

III. Protocol of ISSR PCR using radiolabel

1. Equipment and reagents

a) Thermal cycler

b) Long gel Poly Acrylamide Gel Electrophoresis (manual sequencing) system

c) The reagents including Taq DNA polymerase, 10 x PCR buffer (500 mM KCl, 100 mM Tris-HCl, 0.01% gelatin and 1% Triton X-100), 10 dNTPs stock (1 mM) and 10 ng DNA samples.

d) 1 x TBE (90 mM Tris borate, pH 8.3, 2 mM EDTA).

e) Radiolabelled dATP

2. SSR PCR reaction mix

Template DNA	1 ml (10 ng)
10 x PCR buffer	1 ml (1x)
25mM MgCl₂	0.6 ml (1.5 mM)
1 mM dGTP, dCTP and dTTP each	1 ml X 3 (100 mM)
1 mM dATP	0.75 ml (75 mM)
1 mM a -32p dATP (6000 ci/mmol)	0.25 ml (2 mCi)
4 mM ISSR primer	0.1 ml (0.4 mM)
Taq Polymerase	0.1 ml (0.5 U)
Sterile water	2.3 ml
Total volume	10 ml

3. Thermal Cycling Conditions

Initial denaturation	94^oC for 2 min.

Denaturation	94^oC for 30 sec.	35 cycles
Annealing	50^oC* for 30 sec.
Extension	72^oC for 1 min.

Final extension	72^oC for 10 min.

4. PAGE electrophoresis conditions

Aliquots of amplified DNA from individual PCR reactions should be mixed with formamide stop solution. Four micro-litre of the sample must be denatured at 75⁰C for 2 min., and immediately chilled on ice. Electrophoretic separation must be done on 6% polyacrylamide gel containing 8 M urea in 1 x TBE buffer. After electrophoresis, the gels should be fixed for 2 x 20 min. with 10% glacial acetic acid. The fixed gel must be air-dried and exposed for 4 -12 hrs.

IV. Protocol for Fluorescent ISSR (FISSR) PCR**

1. Equipment and reagents

a) Thermal cycler

b) Automated sequencing System (e.g. ABI 377)

c) The reagents including Taq DNA polymerase (preferably 'AampliTaq Gold' to prevent stutter bands, see Note below), 10 x PCR buffer (500 mM KCl, 100 mM Tris-HCl, and 1% Triton X-100), 10 x each of dGTP, dCTP dTTP and dATP stock (1 mM), 2m M stock of fluorescent dUTP (TAMARA, R110 or R6G, Perkin Elmer) and 5 ng DNA samples.

d) 6 x loading buffer and GENESCAN-1000 ROX- labelled molecular weight standard

e) 1 x TBE (90 mM Tris borate, pH 8.3, 2 mM EDTA)

2. ISSR PCR reaction mix

Template DNA	0.1 ml (5 ng)
10 x PCR buffer	0.5 ml (1X)
25mM MgCl₂	0.3 ml (1.5mM)
1mM dNTPs	0.25 ml (50 mM)
2m M fluorescent dUTP	0.5 ml (0.2 mM)
ISSR primer (4 mM)	0.5 ml (0.4 mM)
Taq Polymerase	0.05 ml (0.25U/ml)
Sterile water	2.30 ml
Total volume	5 ml

3. Thermal Cycling Conditions

Initial denaturation	94^oC for 2 min.

Denaturation	94^oC for 30 sec.	35 cycles
Annealing	50^oC* for 30 sec.
Extension	72^oC for 1 min.

Final extension	72⁰C for 10 min.

4. Sample preparation and electrophoresis conditions

One micro-litre of PCR product should be mixed with 1.5 m l of 6 X loading buffer (1: 4 mixture of loading buffer and formamide; Sigma). To this add 0.4 m l of GENESCAN-1000 ROX- labelled molecular weight standard (red fluorescence). Before loading onto an ABI 377 automated sequencer, the samples should be denatured at 92° C for 1 min. For an optimum separation, denaturing Polyacrylamide gel of 5% containing 7M urea in 1 X TBE buffer must be used.

*NOTE 1: This is a sample reaction condition. Annealing temperature and MgCl₂ concentration varies for each primer set.

**NOTE 2: FISSR-PCR reaction can be performed using fluorescent labelled primers instead of fluorescent dUTP.

NOTE 3: For better results, AmpliTaq Gold (Perkin Elmer) enzyme or any other taq enzyme with anti-taq antibody can be used. Alternatively, manual hot start PCR should be performed where taq is added after initial denaturation step.