SilkSatDB-A Silkworm Microsatellite Database


*SilkSatDb: The First Comprehensive Database on Insect Microsatellites*

Analysis of Simple Sequence Repeats (SSR)

Analysis of Simple Sequence Repeats (SSR)

PRINCIPLE

The Simple Sequence Repeats (SSRs) or microsatellites occur ubiquitously in all the prokaryotic and eukaryotic genomes. Dinucleotide repeats like (CA)_n and (GA)_nare the most abundant repeats in most of the eukaryotes (for eg. in humans (CA)_n repeat occurs once in every 30 kb). Change in number of repeats gives the length polymorphism which is revealed by designing primers for the sequences flanking the microsatellite repeat motif followed by PCR amplification and visualisation in agarose or denaturing polyacrylamide gel. This process of amplification and visualisation can also be automated by labelling the primers with fluorescent dye.

ADVANTAGES

This method requires very small quantity of DNA (~5 ng/reaction) and it is PCR based. This method provides co-dominant and highly reproducible markers that can be readily shared between different labs. The whole microsatellite analysis is automated including multiplexing (analysis of multiple loci in the same lane using different fluorescent labelled primer sets) to enable the researchers to carry out large scale genetic mapping and population studies. Besides being excellent molecular markers for genetic mapping, microsatellite markers are very useful for population genetics, variety identification and protection, monitoring of seed purity and hybrid quality, gene tagging, germplasm evaluation and phylogenetic studies, studies of kinship, conservation genetics and forensics.

LIMITATION
1. Analysis of SSRs requires prior characterisation of sequences flanking the repeats to allow the primer design for PCR amplification, which is experimentally quite labour intensive.

Click on the image to enlarge.

SSR-PCR. Figure showing detection of polymorphism using microsatellite analysis. The arrows represent forward and reverse primers for the (CA)_nrepeats for the same locus. The gel pattern of the amplification products with different combination of alleles is shown in the box

Microsatellites can be analysed using different protocols as per the lab’s budget, labour and speed required. In the past years we have tested many different protocols and standardised them for our population studies, polymorphism estimations, genetic mapping etc., and a list of these protocols is given below.

Protocols List:

Protocol for analysing microsatellites using 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 with 2% difference.

Protocol for analysing microsatellites using 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 use radioactivity but is more labour intensive than agarose gels.

Protocol for analysing microsatellites using radiolabel, is the most sensitive technique among these methods. It is labour intensive and involves the risk of using radioactivity.

Protocol for analysing microsatellites using fluorescent dNTP, is one of the accurate methods for size and band density estimation. This method is automated and thus requires automated sequencing system. It is specially suitable for large scale analysis as multiple loci can be efficiently multiplexed.

I.Protocol for analysing microsatellites using agarose gel electrophoresis

1. Equipment and reagents

a) Thermal cycler

b) Agarose 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) MetaPhor agarose (FMC) and normal agarose

2. SSR PCR reaction mix

Template DNA	1 ml (10 ng)
10 x PCR buffer	1 ml (1x)
MgCl₂(25mM)	0.6 ml (1.5mM)
dNTPs (1mM)	1 ml (100 mM)
Forward primer (4 mM)	1 ml (0.4 m M)
Reverse primer (4 mM)	1 ml (0.4 m M)
Taq Polymerase	0.1 ml (0.5 U)
Sterile water	4.3 ml
Total volume	10 ml

3. Thermal Cycling Conditions

Initial denaturation	94^oC for 2 min.

Denaturation	94^oC for 30 sec.	30 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 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 for analysing microsatellites using 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.

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. SSR PCR reaction mix

Template DNA	1 ml (10 ng)
10 x PCR buffer	1 ml (1x)
MgCl₂(25mM)	0.6 ml (1.5mM)
dNTPs (1mM)	1 ml (100 mM)
Forward primer (4 mM)	1 ml (0.4 m M)
Reverse primer (4 mM)	1 ml (0.4 m M)
Taq Polymerase	0.1 ml (0.5 U)
Sterile water	4.3 ml
Total volume	10 ml

3. Thermal Cycling Conditions

Initial denaturation	94^oC for 2 min.

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

Final extension	72^oC for 10 min.

4. Electrophoresis conditions

Aliquots of amplified DNA from individual PCR reactions should be loaded on a denaturing 6% Poly Acrylamide Gel containing 7M urea in 1x TBE. After the bromophenol blue dye runs out, the gel must be 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. The stained gels are dried at room temperature and stored in photographic albums.

III. Protocol for analysing microsatellites using radiolabel

1. Equipment and reagents

a) Thermal cycler

b) Long gel Poly Acrylamide Gel Electrophoresis system

c) Reagents including Taq DNA polymerase (preferably 'AmpliTaq 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), a -32p dATP and 20 ng DNA samples.

d) Stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% Xyline cyanol in the ratio of 3:2).

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

2. SSR PCR reaction mix

Template DNA	2 ml (20 ng)
10 x PCR buffer	2 ml (1x)
Upper primer (4 mM)	2 ml (0.4 mM)
Lower primer (4 mM)	2 ml (0.4 mM)
dGTP, dCTP and dTTP each (1 mM)	2 ml X 3 (100 mM)
dATP (1 mM)	0.5 ml
a -32p dATP (1000 ci/mmol)	0.4 ml
MgCl₂(25mM)	1.2 ml (1.5 mM)
Taq Polymerase	0.2 ml (1U)
Sterile water	3.7 ml
Total volume	20 ml

3. Thermal Cycling Conditions

Initial denaturation	94⁰C for 2 min.

Denaturation	94^oC for 30 sec.	30 cycles
Annealing	50^oC* for 30 sec.
Extension	72^oC for 45 sec.

Final extension	72^oC for 10 min.

4. 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 95^oC 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 analysing microsatellites using fluorescent dNTP**

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. SSR 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	0.5 ml (100 mM)
2m M fluorescent dUTP	0.5 ml (0.2 mM)
Upper primer (4 mM)	0.5 ml (0.4 mM)
Lower primer (4 mM)	0.5 ml (0.4 mM)
Taq Polymerase	0.05 ml (0.25U/ml)
Sterile water	2.05 ml
Total volume	5 ml

3. Thermal Cycling Conditions

Initial denaturation	94^oC for 2 min.

Denaturation	94^oC for 30 sec.	30 cycles
Annealing	50^oC* for 30 sec.
Extension	72^oC for 45 sec.

Final extension	72^oC 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 0.3 m l of GENESCAN-500 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 6M urea in 1 X TBE buffer must be used.

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

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

NOTE 3: To prevent stutter bands (faint bands appearing above and below the actual band because of the repeat structure), AmpliTaq Gold (Perkin Elmer) enzyme or any other taq enzyme with anti-taq antibody can be used. Alternatively, manual hot start PCR could be performed where taq polymerase is added after initial denaturation.