Molecular Markers RFLP, RAPD, AFLP, STS ,SCAR ,SSCP& QTL -INTRODUCTION -PRINCIPLE -PROCERDURE -APPLICATION -ADVANTAGES -DISADVANTAGES

1. RESTRICTION FRAGMENT LENGTH POLYMORPHISM
R F L P
ALEC JEFFERYS

2. RFLP was the first DNA profiling technique inexpensive enough to
see widespread application.
PRINCIPLE
• RFLP is an enzymatic procedure for separation and identification
of desires fragments of DNA.
• Using restriction endonuclease enzymes fragments of DNA is
obtained and the desired fragments is detected by using restriction
probes.
• It is used to differentiate between the two organisms by analysis of
patterns.

3. PROCEDURE

4. EXAMPLE

5. RESULT:
Crime
Suspected
Suspected
01
Suspected
02

6. Forensic Sciences
Parenting Test
Gene Mapping Studies
Risk Analysis For Genetic Diseases

7. DISADVANTAGES
ADVANTAGES
 Measure variation at the level of DNA
sequence.
 Gene mapping.
 Criminal identification.
 Co-dominant.
 Phylogenetic relationship.
 Paternity test.
 Requires large amount of genomic
DNA.
 Not useful in detecting point
mutation.
 Level of polymorphism is low.
 RFLP is a slow process.
 It involves southern blotting.
 Uses radioactive probes that are
hazardous.
 Requires expertise.

8. R A P D
Random Amplified Polymorphic DNA
Williams & Welsh and McClelland, 1990

9. INTRODUCTION
• RAPD markers are decamer DNA fragments
• RAPD is a type of PCR reaction, segments amplified are
random.
• No knowledge of DNA sequence required, Hence a
popular method.
• In recent years, RAPD is used to characterize, & Trace,
the phylogeny of diverse plant & animal species.
• Identical 10-mer primer will or will not amplify a
segment of DNA, depending on positions that are
complementary to the primer sequence.

10. PRINCIPLE:
RAPD is a type of polymerase chain reaction (PCR) , but the
segments of DNA that are amplified in this process are random.
Specific portion of DNA Randomly

11. As the temp increases 95c there
will be DENATURATION
Amplification takes place
(closed enough or near to)
Amplification does not
takes place
(far apart)
ATTENUATION
PROCEDURE

12. EXAMPLE:

13. USES OF
RAPD
Forensic sciences
Crops in variety
identification
Microbial strain determination
Gene mapping studies

15. A F L P
AMPLIFIED FRAGMENT LENGTH POLYMORPHISM
Pieter Vos and his colleagues in 1995

16. PRINCIPLE:
Amplified fragment length polymorphism (AFLP) is a PCR-based
technique that uses selective amplification of a subset of digested
DNA fragments to generate and compare unique fingerprints for
genomes of interest.

17. PROCEDURE

18. USES OF AFLP
FORENSIC SCIENCES
GENETIC VARIATION
CRIMINAL INVESTIGATION
CROP VARIETY
PATERNITY TEST

20. Olson (1989)

21. STS is a relatively short, easily PCR-
amplified sequence (200 to 500 bp)
which can be specifically amplified
by PCR and detected in the presence
of all other genomic sequences and
whose location in the genome is
mapped.
• STS derived from a region
cDNA.
• STS occurs only once in genome.
• Both the ends of the site are
unique.
• STS includes such markers as
microsatellites SCAR & SSCP.

22. USES OF STS
Detecting Microdeletions
Identification of genes in
elephants
Evolutionary Studies
Screening PCR to
detect microdeletions in
azoospermia genes in
infertile men

23. ADVANTAGES DISADVANTAGES
• This technique permits
sharing of data across the
laboratories.
• It has high degree of
accuracy.
• Codominant.
• Target specific genes.
• Highly reproducibility.
• Development of STS is a
difficult task.
• It is time consuming to develop.
• It required high technical skills.
• Needs prior sequence
information.

24. SEQUENCE CHARACTERIZED AMPLIFIED REGION
S C A R

25. INTRODUCTION
PRINCIPLE:
Conversion of RAPD or AFLP polymorphism into locus
specific marker.
 SCAR marker is based on sequence of polymorphic bands from
RAPD/RFLP/AFLP linked to trait of interest.
 Longer primers (15-30bp) are designed for specific
amplification of particular locus.
 SCAR markers are delimited by the sequence of the primers
and they can be both dominant (presence/absence of a given
band) and co-dominant (bands with different sizes in different
samples), and usually they are considered single locus.

27. USES OF SCAR
• Identification of sex types in plantlets
growing in field. During the cultivation
of dioecious plant sex determination is
more useful and profitable. E.g. papaya
species have three sex types (male,
female and hermaphrodite)
• Allow the amplification of specific
sequences on well-known loci,
previously identified by other
markers . Such as AFLP RAPD.
• Biodiversity studies.

28. • Allow the amplification of specific
sequences on well known loci,
previously identified by other
markers such as AFLP, RAPD,SSR
markers.
• Dominant.
• High reproducible.
• Time consuming to develop.
• Needs prior sequence
information.
• Relatively few in number.
ADVANTAGES DISADVANTAGES

29. S S C P
SINGLE STRAND CONFORMATION POLYMORPHISM

30. PRINCIPLE:
• SSCP allows identification of different genomic variants in a
large number of samples & in a broad range of organisms.
• A sensitive method of mutant detection without sequencing.
• Based on single-strand DNA has defined conformation.
• DNA with a single base variation migrate differently and
shows different banding pattern under non-denaturing
condition.
DNA samples are subjected to gel electrophoresis in non-
denaturing conditions.

31. PROCEDURE:

32. USES OF SSCP
Detect Mutation
Diagnostic Tools
Detect Various Strains
in Viruses
Large number of human
disease genes as in tumor

33. ADVANTAGES DISADVANTAGES
• The genes contain sufficient
polymorphism.
• Which of the gene is most
polymorphic.
• May individual PCR products
are screened simultaneously.
• High reproducibility.
 Absence of mutation cannot be
proven, since some mutation may
remain undetected.
 Sequence data needed for primer
construction.
 High standardized electrophoretic
condition is needed.
 Time consuming

34. Q T L
QUANTITATIVE TRAIT LOCI
Lander and Botstein (1989)

35. INTRODUCTION
• A QTL is defined as “a region of the
genome that is associated with an effect
of a quantitative trait.” So a QTL can be a
single gene, or it may be a cluster of
linked genes that affect the traits.
• QTL mapping studies have reported in
most of the crop QTL MAPPING 14
plants for diverse traits like yield, quality
disease and insect pest resistance, abiotic
stress tolerance and environmental
adaptation.
PRINCIPLE
calculated for a given set of parameters (particularly QTL effect and
QTL position) given the observed data on phenotypes and marker
genotypes.

36. PROCEDURE:

37. ADVANTAGES
 QTL analysis is to provide insights
into whether differences of
phenotype are primarily affected by
a few loci with large effects, or by
numerous loci, each with tiny
effects.
DISADVANTAGES
 You are limited to the genetic diversity
present into the parents of your
segregating population.
 QTL studies require very large sample
sizes
QTL analysis allows researchers in fields as diverse as
agriculture, evolution, and medicine to link certain complex
phenotypes to specific regions of chromosomes.
APPLICATION