Microsatellite

Dr Karan Veer Singh

NBFGR

What is a microsatellite?
Tandemly repeated DNA (may see in the
literature as STRs - Short tandem repeats)
Poly A/T most common
1-10 bp tandemly repeated = micro satellite
>10 = mini satellite

Types of microsats

Di, tetra and tri nucleotide (used in that order)

Perfect
Imperfect/interrupted
Compound
Varying levels of variation associated with each type
Difficulty in scoring

Short Tandem Repeats (STRs)

AATG

7 repeats
8 repeats
the repeat region is variable between samples while the
flanking regions where PCR primers bind are constant
Homozygote

= both alleles are the same length

Heterozygote = alleles differ and can be resolved

from one another

 Also called as STR, SSR, VNTR

Tandemly repeated DNA sequences with the
repeat/size of 1 6 bases repeated several
times
Highly polymorphic; can be analysed with the
help of PCR
Individual alleles at a locus differ in number
of tandem repeats of unit sequence owing to
gain of loss of one or more repeats and they
can be differentiated by electrophoresis
according to their size
Powerful DNA markers for quantifying genetic
variations within & between populations of a
species

Microsatellites Types
Based on repeat pattern
1. Perfect

CACACACACACACACACACACA

2. Imperfect

CACACACACA
CACACACA

CACACACA

3. Compound
CACACACACACACA CATACATACATA CATACATACATA

4. Complex

CACACACACACACACA
AATAATAATAATAATAATAAT

Based on number of base pairs

1) Mono (e.g. CCCCCCCC or AAAAAA)
2) Di (e.g. CACACACACA)
3) Tri (e.g. CCA CCA CCA CCA)
4) Tetra (e.g. GATA GATA GATA GATA GATA GATA GATA)

Minisatellites: - (9 65 base pairs repeated from 2

to several hundred times)
CGCCATTGTAGCCAATCCGGGTGCGATTGCAT CGCCATTGT
AGCCAATCCGGGTGCGATTGCAT
TGCGATTGCAT

CGCCATTGTAGCCAATCCGGG

CGCCATTGTAGCCAATCCGGGTGCGATTGCAT

CGCCATTGTAGCCAATCCGGGTGCGATTGCAT

Microsatellites Properties
Co-dominant

Inherit in Mendelian Fashion

Polymorphic loci with allele number as high as 14 15
per locus
Mostly reported from non-coding region, hence can be
independent of selection
Flanking region is highly conserved in related species
Can be obtained from small amounts of tissues [STR
analysis can be done on less than one billionth of a
gram (a nanogram) of DNA (as in a single flake of
dandruff)]
PAGE separation; silver staining/automated genotyping
Abundant in the eukaryote genome (~103 to 105 loci
dispersed at 7 to 10100 kilobase pair (kb) intervals)

Microsatellites and Human Diseases

Allele size variations in microsatellite loci in close
proximity (showing linkage disequilibrium) to the following
genes within the Human Major Histocompatibility Complex
(MHC) region

IDDM (Insulin Dependent Diabetes Mellitus)

Multiple Sclerosis (MS)

Narcolepsy

Uveitis

have been reported to cause genetic disorders; hence these

genetic disorders can be detected by screening the allele
sizes of the microsatellite loci (Type I markers) that are in
close proximity to these genes (Goldstein & Schlotterer,
2001)

The microsatellite, or short sequence repeat (SSR), is a

powerful genetic marker, useful in many areas of fish genetics
and breeding.
Polymorphic microsatellite loci have been frequently applied
to the analysis of genetic diversity,
population genetic structure, and
genomic mapping.
These co-dominant markers have also been applied to the
classification and systematics,
parentage identification,
germplasm conservation, and
breeding programme of food fish.

zebrafish

The
is the first vertebrate organism used for largescale genetic screens seeking genes critical to development.
1800 recessive mutations discovered to morphogenesis of the
vertebrate embryo. The cloning of the mutant genes depends on a
dense genetic map.

The 2000 markers, using microsatellite

provides 1.2-cM average resolution.

(CA) repeats,

One centimorgan in zebrafish is about 0.74 megabase, so, for many

mutations, these markers are close enough to begin positional cloning
by YAC walks.

A Microsatellite Genetic Map of the Turbot (Scophthalmus maximus)

Genetics. 2007 December; 177(4): 24572467.

A consensus microsatellite-based linkage map was constructed from two

unrelated families. The mapping panel was derived from a gynogenetic family
of 96 haploid embryos and a biparental diploid family of 85 full-sib progeny
with known linkage phase.
A total of 242 microsatellites were mapped in 26 linkage groups.
The consensus map length was 1343.2 cM, with an average distance between
markers of 6.5 0.5 cM.

The comparison of turbot microsatellite flanking sequences against

the Tetraodon nigroviridis genome revealed 55 significant matches, with a
mean length of 102 bp and high sequence similarity (81100%).
This map will be suitable for QTL identification of productive traits in this
species and for further evolutionary studies in fish and vertebrate species.

Cloning and isolating genes:

Locating a gene is easy if the gene product (protein) is identified.

1.

Create a cDNA library using an expression vector.

2.

Probe with antibodies that bind the gene product.

3.

Isolate and sequence positive clones.

If the gene product is unknown, locating a gene is more difficult.

1.

Identify a marker (microsatellite, RFLP, SNPs) that is physically linked to

the gene on the same chromosome, and segregates in test crosses with the
disease phenotype (i.e., shows a strong statistical association).

2.

Use a technique called positional cloning to home in on gene.

e.g., cloning and discovery of the cystic fibrosis (CF) gene.

Markers Search
1. Type marker name.
Use * for wildcards.

2. To refine search, specify a

marker type and/or a taxonomy
(species). For taxonomy use
common or scientific name.

3. Click search
icon.
Or Click to run sample
searches.

Clicking on Search
lets you search for
markers of that type.
Clicking on a marker type lets
you view a definition of that
marker type.

These tables show a

breakdown of markers
in the database by
marker type.

Genetic Diversity Studies of seahorse: Microsatellite

3.Molecular Data Analysis using Genetic software

Sequence editing /Processing/ Submission
Molecular data analysis (EditSeq, MegaBACE, CLUSTALW,BIOEDIT, MEGA 4, Arlequin)
Phylogenetic analysis (PAUP,MOLPHY,MEGA,AMOVA)

Sites of occurrences

Total 23 alleles

No Gene Flow
Genetic Tags

Stock specific Markers

Partitioning of Breeding Population
Limitation in Migration

Total 11 Private Alleles

No Mixing of Gene Pool

* Stock- Specific markers

* Genetic TAGs for selection programs

Microsatellites PCR reaction Mixture &

Concentration

Volume per
reaction

Double distilled water

18.0L

Assay buffer (10X; Genei, Bangalore, India)

(100mM Tris, 500mM KCl, 0.1% gelatin, pH9) (final
conc. 1X)

2.5L

dNTPs (Genei, Bangalore, India) (200 mM)

2.0L

Primer (forward & reverse working solution) (total

conc. ~ 10.0 pmoles in 25l of master mix)

0.5L

MgCl2

0.5 l

(1.5mM )

Taq polymerase (Genei, Bangalore, India) (3Units/

l)

0.5L

Template DNA (25ng)

1.0L

Total volume

25.0L

Microsatellite PCR

10% non- denaturing Polyacrylamide gel

electrophoresis (PAGE) to separate the PCR
products

Acrylamide (19:1 acrylamide

and bisacrylamide)

5mL

Double distilled water

2mL

5 x TBE

2mL

10% Ammonium persulphate

70L

TEMED

3.5L

M 1

3 4

9 10 11 ve M

Single Locus Microsatellites

(PAGE & Silver staining;
There are five alleles at this locus)
M: Standard molecular weight marker (pBR322 DNA/MspI digest).
1 -11: Different individuals -ve : Negative control

Microsatellites- multiplexing &

Use of Fluorescent dyes

Automated Genotyping

A sample print-out for one person, showing all 16 loci tested.

Different colours help with interpretation

Thanks
The Improbable seahorses,National Geography 1994