- Molecular markers are segments of DNA that represent genetic differences and can be used for genetic analysis, though they may not correlate with observable traits. - An ideal molecular marker technique should be polymorphic, provide adequate genetic resolution, generate multiple independent markers simply and inexpensively using small amounts of DNA, and be linked to distinct phenotypes. - Molecular marker techniques can be categorized as non-PCR based like RFLP analysis or PCR-based like AFLP, RAPD, and SNP analysis, which have been widely used in plant and animal research.

1. WELCOME

2. ADVANCED MOLECULAR
MARKERS

3. A molecular marker:-
• segment of DNA that is representative of the differences at
the genome level
• may or may not correlate with phenotypic expression of a
trait
• are stable and detectable in all tissues regardless of growth,
differentiation, development

4. An ideal molecular marker technique should
have the following criteria
• (1) be polymorphic and evenly distributed throughout the genome,
• (2) provide adequate resolution of genetic differences
• (3) generate multiple, independent and reliable markers
• (4) simple, quick and inexpensive
• (5) need small amounts of tissue and DNA samples;
• (6) have linkage to distinct phenotypes and
• (7) require no prior information about the genome of an organism.

5. Molecular marker techniques:-
• two categories:
• (1) non-PCR-based techniques or hybridization based techniques
• Restriction fragment length polymorphism (RFLP)
• (2) PCR-based techniques.
• AFLP,RAPD,SNP etc

6. TECHNIQUE
• The resulting DNA fragments are then separated by length
throughAGE
• Southern blot
• Hybridization
• determines the length of the fragments.
• An RFLP occurs when the length of a detected fragment varies
between individuals
• Each fragment length is considered an allele, and can be used
in genetic analysis.

7. How RFLP Pattern differs in different
organisms
• Mutations can add /delete R.Sites

8. RFLP analysis may be subdivided into
• single- (SLP)
• more sensitive
• easier to interpret and
• capable of analyzing mixed-DNA samples .
• data can be generated even when the DNA is degraded (e.g. when it is found
in bone remains.)
• multi-locus probe (MLP) paradigms.

9. Multilocus Vs Single Locus probes

10. • Allele A
• a small segment of the genome is being detected by a DNA probe
(thicker line). In allele "A",
• the genome is cleaved by a restriction enzyme at 3 nearby sites
(triangles),
• but only the rightmost fragment will be detected by the probe.
• In allele "a",
• restriction site 2 has been lost by a mutation,
• the probe now detects the larger fused fragment running from sites 1
to 3.

11. • In allele "c" ------------5 repeats in the VNTR,
• the probe detects a longer fragment between the two restriction sites.
• In allele "d" ----------- only 2 repeats in the VNTR,
• the probe detects a shorter fragment between the same two restriction sites

12. • TRFLP
• PCR amplification of DNA using fluorescenly labelled primer pairs
• The PCR products are then digested using RFLP enzymes
• patterns visualized using a DNA sequencer.
• results are analyzed either by simply counting and comparing bands or peaks
in the TRFLP profile, or by matching bands from one or more TRFLP runs to a
database of known species.
• The technique is similar in some aspects to DGGE or TGGE.

13. • TRFLP
• PCR amplification of DNA using
fluorescenly labelled primer pairs
• The PCR products are then digested
• patterns visualized using a DNA
sequencer.
• results are analyzed by simply
counting and comparing bands or
peaks in the TRFLP profile,.
• The technique is similar in some
aspects to DGGE or TGGE.

14. II) PCR-based techniques

15. RAPD/ Random Amplified Polymorphic DNA
• a type of PCR reaction,
• but the segments of DNA that are amplified are random
• Uses several arbitrary, short primers (8–12 Nt.),
• then proceeds with the PCR using a large template of genomic DNA,
• hoping that fragments will amplify.
• By resolving the resulting patterns, a semi-unique profile can be
gleaned from a RAPD reaction.
• inexpensive

16. • the primers will bind somewhere
in the sequence
• to characterize, and trace,
the phylogeny of diverse plant
and animal species.
• is used to analyse the genetic
diversity of an individual by
using random primers.

17. Amplified fragment length polymorphism (AFLP)
• To overcome the limitation of reproducibility associated with RAPD,
• It combines the power of RFLP with the PCR
• ligating primer recognition sequences (adaptors) to the restricted DNA
• selective PCR amplification of restriction fragments using a limited set of
primers
• usually produce 50–100 bands per assay
• generates fingerprints of any DNA regardless of its source, and without any
prior knowledge of DNA sequence.
• can be used to distinguish closely related individuals at the sub-species level
• in plant mapping
• fluorescence tagged primers are also used

18. ADAPTORS

19. ADAPTORS

20. LINKERS

21. PROCEDURE:-

22. SCAR
• utilize markers identified by arbitrary marker analysis (RAPD, AFLP,
etc.
• cloning the amplified products
• then sequencing the two ends of the cloned products.
• The sequence is thereafter used to design specific primer pairs of 15–
30 bp which amplify single major bands of the size similar to that of
cloned fragment
• SCARs are primarily defined genetically, they can be used both as
physical landmarks in the genome and as genetic markers.

23. Sequence characterized amplified regions
(SCAR)

25. Cleaved Amplified Polymorphic Sequences
(CAPS)

26. CAPS:-

27. CAPS:-

28. Advantages of CAPS
• Most CAPS markers are
co-dominant and locus-
specific.
• Most CAPS genotypes are
easily scored and
interpreted.
• CAPS markers are easily
shared between
laboratories.
• CAPS assay does not
require the use of
radioactive isotopes, and
it is more amenable,
therefore, to analyses in
clinical settings.

29. Developing CAPS markers
• Sequence the RFLP probe.
• Design primers to amplify 800–2,000-bp DNA fragments. Targeting
introns or 3' untranslated regions should increase the chance of
finding polymorphisms
• The PCR product is cloned and sequenced.
• PCR amplify DNA fragments from target genotypes, separately digest
the amplicons with one or more restriction enzymes.
• Screen the digested amplicons for polymorphism on gels stained with
ethidium bromide

30. Randomly amplified microsatellite
polymorphisms (RAMP
• show a high degree of allelic polymorphism
• radiolabeled primer consisting of a 5’ anchor and 3’ repeats
• Amplify genomic DNA in the presence/absence of RAPD primers.
• PAGE/AGE
•
5’ANCHOR-ieCGATA Repeat-ie-GCGCGCGC3'
3’GCTAT CGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCG5’

31. RAMP Primer
• The melting temperatures of the anchored primers are usually 10–
15oC higher than those of the RAPD primers
• at higher annealing temperature
• only the anchored primer would anneal efficiently,
• in PCR cycles at low annealing temperature
• both anchored microsatellite and RAPD primers would anneal.

32. • Most fragments obtained with RAMP primers alone disappear when
RAPD primers are included, and different patterns are obtained with
the same RAMP primer and different RAPDs, indicating that RAPD
primers compete with RAMP primer during the low annealing
temperature cycle.

33. At low
Annealing
Temp
At High
annealing
Temperatur
e
Both RAMP
&RAPD
primer
anneals
Only RAMP
primer
anneals
Bands of
both
primers
only RAMP
primer
bands
Ie-RAPD primers can
successfully compete
with the RAMP
primers in the
amplification reaction

35. Sequence-related amplified polymorphism
(SRAP)
• to amplification of open reading frames (ORFs)
• uses primers of arbitrary sequence,
• 17–21 nucleotides in length
• It uses pairs of primers with AT- or GC- rich cores to amplify intragenic
fragments for polymorphism detection.

36. SRAP:-
• The primers consist of the following elements
• 1. Filler sequences,
• 13–14 bases long,
• where the first 10 or 11 bases starting at the 5’-end, are sequences of no specific
constitution (‘‘filler’’ sequences),
• The filler sequences of the forward and reverse primers must be different from each
other
• 2. The core sequence:-
• followed by the sequence CCGG in the forward primer and AATT in the reverse primer
• is followed by three selective nucleotides at the 3’-end.

37. SRAP:-
PCR
1st 5cycles annealing temp-35oC
Followed by 35 cycles temp-50oC
PAGE
Autoradiography

38. SRAP:-
• fragment size changes due to insertions and deletions-CODOMINANT
• nucleotide changes leading to ---------------------DOMINANT marker

39. Target region amplification polymorphism (TRAP)
• The technique uses two primers (18 nucleotides in length) to
generate markers.
• One of the primers, the fixed primer,
• is designed from the targeted EST sequence in the database;
• the second primer
• is an arbitrary primer with either an AT- or GC-rich core to anneal with an intron or exon.

40. THANK YOU