Retrotransposon-basedmarkers (S-SAP, IRAP, REMAP and RBIP)

1. Retrotransposon-based markers
(S-SAP, IRAP, REMAP and RBIP)
Farrokhzadyusuf@gmail.com
Yusuf Farrokhzad
1/28/2018 1

2.  In 1940s Barbara McClintock did a series
of genetic experiments with corn that led
her to hypothesize the existence of what
she called the “controlling genes” which
modify or suppress gene activity and are
mobile in the genome (1950).
A Brief History:
 Barbara Mc Clintock 1902 -1992 ( noble in 1984)
 She spent the three decades for this genetic elements.
1/28/2018 2

3. We now know that only 1.1% to 1.4% of our DNA actually encodes proteins.
More than 50% of our genome
consists of short, repeated
sequences, the vast majority of
which—about 45% of our
genome in all—come from
transposons.
High proportion of species genome:
 10% of several fish species
 12 % of the Caenorhabditis. elegans
genome
 37% of the mouse genome
 45% human genome
 up to >80% - some plants like maize
1/28/2018 3

4. 1. Transposon: a DNA sequence with the ability to
move and be inserted at a new location of the
genome.
2. Retrotransposon :a mobile DNA sequence that
can insert itself at a different position by using
reverse transcriptase.
 Normal ubiquitous components of the genomes
 Jumping genes
 Selfish DNAs
 Molecular parasites
 Controlling elements
 They can cause genome rearrangement (mutation,
deletion and insertion).
They have wide
range of
application
potentials They are the major forces driving evolution
1/28/2018 4

5. More details:
 More abundant in eukaryotic genomes than prokaryotes
Transposons are a major source of genetic variation.
This lends the genome flexibility to adapt to
changing environmental conditions during the
course of evolution. Although the precision of the
genetic information depends on stability, complete
stability would also mean static persistence. This
would be detrimental to the development of new
forms of life. Genomes are subject to alterations, as
life requires a balance between the old and the new.
1/28/2018 5

6. Classification of Transposable Elements
Transposons can be divided into the following three
families on the basis of their overall organization and
mechanism of transposition.
1) DNA transposons: Encodes proteins that moves the DNA element directly to a new position or
replicate the DNA to produce a new element that integrates elsewhere in the genome (both
prokaryotes and eukaryotes).
2)Virus-like retrotransposons: These elements are also called long terminal repeat (LTR) retrotransposons.
Encodes a reverse transcriptase for making DNA copies of their RNA transcripts which subsequently integrates
at new sites in the genome (only prokaryotes)
3) Poly-A retrotransposons: These elements are also called non-viral retrotransposons. The element terminates
in the 5’ and 3’ untranslated region (UTR) sequences and encodes two enzymes: an RNA-binding enzyme
(ORF1) and an enzyme having both reverse transcriptase and endonuclease activities (ORF2).
1/28/2018 6

7. 1/28/2018 7

8. Plant genomes are rich in transposons:
Maize color varigation due to chromosome
breakage by transposition
Snapdragons: size of white patches related
to frequency of transposition

9. The cut-and-paste mechanism of
transposition. Movement of a
transposon from a target site in the
host DNA to a new site in the DNA.
1/28/2018 9

10. 1/28/2018 10

11. o Retrotransposon
Because retrotransposon insertions are irreversible, they are
considered particularly useful in phylogenetic studies. In addition,
their widespread occurrence throughout the genome can be exploited
in gene mapping studies, and they are frequently observed in regions
adjacent to known plant genes.
o Inter-Retrotransposon amplified polymorphism (IRAP):
o Sequence-specific amplification polymorphism (S-SAP):
o Retrotransposon-microsatellites amplification polymorphism (REMAP):
o Retrotransposon-based amplification polymorphism (RBIP):
1/28/2018 11

12. o Sequence-specific amplification polymorphism (S-SAP):
Sequence-Specific Amplified Polymorphism (S-SAP) is a
dominant, multiplex marker system for the detection of
variation in DNA flanking the retrotransposon insertion site.
Retrotransposon containing fragments are amplified by PCR,
using one primer designed from the conserved terminus of
the LTR and one based on the presence of a nearby
restriction endonuclease site.
Experimental procedures resemble those used for AFLP
analysis and they are usually dominant markers. Compared
to AFLP, S-SAP generally yields fewer fragments but higher
levels of polymorphism.
1/28/2018 12

13. 1/28/2018 13

14. Inter-Retrotransposon Amplified Polymorphism (IRAP) are dominant, multiplex marker
systems that examine variation in retrotransposon insertion sites.
o Inter-Retrotransposon amplified polymorphism (IRAP):
With IRAP, fragments between two retrotransposons are isolated by PCR, using outward-
facing primers annealing to LTR target sequences.
IRAP as well as REMAP fragments can be
separated by high-resolution agarose gel
electrophoresis.
1/28/2018 14

15. o Retrotransposon-microsatellites amplification polymorphism (REMAP):
Retrotransposon-Microsatellite Amplified Polymorphism (REMAP) are
dominant, multiplex marker systems that examine variation in
retrotransposon insertion sites.
In the case of REMAP, fragments between retrotransposons and
microsatellites are amplified by PCR, using one primer based on a LTR
target sequence and one based on a simple sequence repeat motif.
IRAP and REMAP fragments can be separated by high-resolution
agarose gel electrophoresis.
1/28/2018 15

16. 1/28/2018 16

17. Retrotransposon-Based Insertional Polymorphism (RBIP) is a
codominant marker system that uses PCR primers designed
from the retrotransposon and its flanking DNA to examine
insertional polymorphisms for individual retrotransposons.
o Retrotransposon-based amplification polymorphism (RBIP):
Presence or absence of insertion is investigated by two PCRs,
the first using one primer from the retrotransposon and one
from the flanking DNA, the second using primers designed
from both flanking regions.
Polymorphisms are detected by simple agarose gel
electrophoresis or by dot hybridization assays. A drawback of
the method is that sequence data of the flanking regions is
required for primer design.
1/28/2018 17

18. 1/28/2018 18

19. 1/28/2018 19

20. 1/28/2018 20

21. 1/28/2018 21

22. THANK YOU…
1/28/2018 22