This document discusses molecular markers, which are short DNA sequences that reveal polymorphisms between individuals. Molecular markers include SNPs, insertions/deletions, and variations in simple sequence repeats. The document describes several types of molecular markers and methods used to detect them, including RFLPs, RAPDs, AFLPs, SSRs, ISSRs, and CAPS. The applications of molecular markers include paternity testing, disease diagnosis, forensic analysis, and varietal identification in plants.

1. MOLECULAR MARKERS
Dr. Muhammad Arif
Genomics, Proteomics and Bioinformatics

2. Overview
• Definition of molecular markers
• Polymorphism
• Causes of polymorphism
• Types of molecular markers
• Methodology of molecular markers
• Applications
11/30/2020 NIBGE Ph.D lecture

3. Molecular markers
Molecular markers are defined as the short
sequences of DNA (primers or probe) which
directly reveal the polymorphism of DNA.
The polymorphic sequences correspond or not
to coding sequences.

4. What is Polymorphism ?
- Variation/difference in
the arrangement of the
nitrgoenous bases
(A,T,G,C) in the DNA of
an individual which is
different from another
individual of the same
specie at a particular
location - May or may
not cause change in
phenotype

5. DNA Variation
Single nucleotide

6. Single Nucleotide polymorphism (SNP)
6
SNPs are the most common type of genetic variation among individuals

7. DNA Variation
Simple Seqeunce Repeat

8. Causes of Polymorphism
1- Insertion or deletion
at or between restriction sites
or
PCR primer sites
2- Changes in the number of repeat unit
between restriction sites or
PCR primer sites
3- Mutations at single nucleotides: SNP

9. Types of Molecular Markers
– Based on DNA-DNA hybridization, e.g.
RFLP
– Based on PCR
• Using random primers:
RAPD, DAF, AP-PCR, ISSR
• Using specific primers:
SSR, SCAR, STS
– Based on PCR & restriction cutting:
AFLP, CAPS
– Based on DNA point mutations (SNP),
can be detected by
SSCP, sequencing,

10. RFLPs are differences in restriction
fragment lengths caused by SNPs or
INDELs that create or abolish restriction
endonuclease recognition sites.
RFLP assays are performed by hybridizing
a chemically labelled DNA probe to a
Southern blot of DNA digested with a
restriction endonuclease.
Restriction Fragment Length Polymorphisms
(RFLPs)

11. RFLPs
• Restriction fragment
length polymorphism
• Co-dominant
• Requires:
single copy DNA probe
Restriction enzyme
Southern blotting
DNA polymorphism
Individual 1
Individual 2

12. Randomly Amplified Polymorphic DNA
RAPDs
• Based on a 10 bp
single arbitrary
primer
• Cheap, easy
• Insufficient
reproducible
maize lines; only primer 2 and 5
demonstrate polymorphism

13. RAPD – dominant marker
A
B

14. •A combination of PCR and RFLP
•Informative fingerprints of amplified
fragments
Amplified Fragment Length Polymorphisms
AFLPs

15. AFLP: Major Steps
• Restriction endonuclease digestion of genomic
DNA and ligation of specific adapters
• Amplification of the restriction fragments by PCR
using primer pairs containing common sequences
of the adapter and two or three arbitrary
Nucleotides
• Analysis of the amplified fragments using gel
electrophoresis

16. AFLP-Major Steps
• Genomic DNA double digests with a 4-cutter
(MseI) and a 6-cutter (EcoR1)
• Ligate adapters to the EcoR1 and MseI RE sites
• Primers complementary to Adapters with selective
nucleotides at 3’ ends and perform PCR
amplification
• Separate DNA fragments on high-resolution gels
• After detection, screen for band polymorphisms

17. AFLP: Restriction and Ligation to Adapters

18. AFLP: Pre-Selective Amplification
Primer (+ 1) for pre-selective amplification

19. AFLP: Selective Amplification
Primer (+ 3) for selective amplification

21. AFLP – band polymorphisms

22. • Simple sequence repeats (SSRs) or microsatellites
are tandemly repeated mono-,di-, tri-, tetra-, penta-,
and hexa-nucleotide motifs.
SSR length polymorphisms are caused by differences
in the number of repeats.
• SSR loci are “individually amplified by PCR using
pairs of oligonucleotide primers specific to unique DNA
sequences flanking the SSR sequence”.
Simple Sequence Repeats
(SSRs)

23. Why Have SSRs Had Such a Large Impact
on Genomics?
• SSRs tend to be highly polymorphic.
• SSRs are highly abundant and randomly dispersed
throughout most genomes.
• Most SSR markers are co-dominant and locus
specific.
• Genotyping throughput is high and can be
automated.

24. SSR - methodolgy
Genotype A
Genotype A Genotype B
PCR amplification with
radiolabelled nucleoltide
Polyacrylamide Gel Electrophoresis
of PCR products and autoradiography
[AT]18 [AT]22
[AT]
18
[AT]
22
Genotype B

25. SSRs display multiple alleles

26. • No sequence knowledge is required
• Primers based on a repeat sequence with a
degenerate 3’ or 5’ anchor, e.g.
CACACACACACACACARG or
AGCAGCAGCAGCAGCAGCTY
• Good for determination of closely
related individuals
Inter Simple Sequence Repeats
(ISSR)

28. • Polymorphisms-----differences in restriction fragment
lengths caused by SNPs or INDELs
• Assays are performed by digesting locus-specific PCR
amplicons with one or more restriction enzymes
• Can be combined with single strand conformational
polymorphism (SSCP), sequence-characterized amplified
region (SCAR), or random amplified polymorphic DNA
(RAPD) analysis to increase the chance of finding a DNA
polymorphism.
Cleaved Amplified Polymorphic Sequences
(CAPS)

30. • DNA polymorphisms produced by differential
folding of single-stranded DNA with mutations.
• Assays are performed using heat-denatured
DNA on nondenaturing DNA sequencing gels.
•Special gels (e.g., mutation detection
enhancement gels) have been developed to
enhance the discovery of single strand
conformational polymorphisms caused by
INDELs, SNPs, or SSRs.
Single Strand Conformational Polymorphisms
(SSCPs)

32. • Paternity and Maternity Issues
• Criminal Identification and Forensics
• Diagnosis of Inherited Disorders
• Diagnosis of Disease
• Relationships among germplasm
• Varietal purity and identification
• QTL Identification
• Early selection in plants
Applications of DNA Fingerprinting