This document discusses different types of molecular markers used in genetics. It describes markers based on nucleic acid hybridization, such as restriction fragment length polymorphism (RFLP), which involves isolating DNA, digesting it with restriction enzymes, separating fragments via electrophoresis, and hybridizing with probes. It also describes polymerase chain reaction (PCR)-based markers like random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP), which allow amplification of genomic DNA regions. Sequence characterized amplified regions (SCARs) and cleaved amplified polymorphic sequences (CAPS) are also PCR-based marker techniques discussed.

1. Markers: Based on
hybridization and PCR
Sachin Kumar
B.Sc. Biotech

2. Molecular Markers ?
DNA sequence in the genome which can be located and
identified.
genetic alterations (mutations, insertions, deletions), can
be mapped and identified.
Molecular markers provide a true representations of the
genetic makeup at the DNA level.
They are consistent and not affected by environmental

3. Types
1. Based on nucleic acid (DNA) hybridization (non-PCR
based approaches).
2. Based on PCR amplification (PCR-based approaches).

4. 1. Markers Based On DNA
Hybridization:
The DNA piece can be cloned, and allowed to hybridize
with the genomic DNA which can be detected. Marker-
based DNA hybridization is widely used. The major
limitation of this approach is that it requires large
quantities of DNA and the use of radioactivity (labeled
probes).

5. Restriction fragment length
polymorphism (RFLP):
The procedure basically involves
1. the isolation of genomic DNA,
2. its digestion by restriction enzymes,
3. separation by electrophoresis, and
4. finally hybridization by incubating with cloned and
labeled probes

6. Restriction fragment length
polymorphism (RFLP):

7. 2. Markers Based on PCR
Amplification:
Polymerase chain reaction (PCR) is a novel technique for
the amplification of selected regions of DNA .The
advantage with PCR is that even a minute quantity of
DNA can be amplified. Thus, PCR-based molecular
markers require only a small quantity of DNA to start
with.

8. 1. Random amplified polymorphic
DNA (RAPD);
No knowledge of the DNA sequence for the targeted
gene is required
1. The DNA isolated from the genome is denatured
2. The template molecules are annealed with primers,
3. Amplified by PCR.
Single short oligonucleotide primers (usually a 10-base
primer) can be arbitrarily selected and used for the
amplification DNA segments of the genome.
4. The amplified products are separated on
electrophoresis and identified.

9. 1. Random amplified polymorphic
DNA (RAPD);

10. 2. Amplified fragment length
polymorphism (AFLP):
AFLP is a novel technique involving a combination of
RFLP and RAPD
AFLP is based on the principle of generation of DNA
fragments using restriction enzymes and
oligonucleotide adaptors (or linkers), and their
amplification by PCR.
1. The DNA of the genome is extracted.
2. It is subjected to restriction digestion by two enzymes
(a rare cutter e.g. Msel; a frequent cutter e.g. EcoRI).
3. The cut ends on both sides are then ligated to known
sequences of oligonucleotides

11. 4. Double PCR is performed and DNA is amplified
(PCR primers is radioactively or fluorescently
labeled).
5. Electrophoresis is done to separate the DNA
fragments based on their size and overall negative
charge.
6. Visualization of the band pattern by Autoradiography.
2. Amplified fragment length
polymorphism (AFLP):

12. 2. Amplified fragment length
polymorphism (AFLP):

13. Sequence characterized amplified
regions (SCARs):
Sequence tagged sites (STS) represent unique simple
copy segments of genomes, whose DNA sequences are
known, and which can be amplified by using PCR.
SCARs are the modified forms of STS markers. They
are developed by PCR primer that are made for the ends
of RAPD fragment. The STS-converted RAPD markers
are sometimes referred to as SCARs. SCARs are useful
for the rapid development of STS markers.

14. CAP(Cleaved Amplified Polymorphic
Sequence)
The cleaved amplified polymorphic
sequence or CAPS method is a technique in molecular
biology for the analysis of genetic markers.
It is an extension to the Restriction Fragment Length
Polymorphism (RFLP) method, using polymerase chain
reaction (PCR) to more quickly analyse the results.
It is difference in restriction fragment length caused by
SNP that can create or abolish the recognition site.

15. Thank you