MOLECULAR MARKERS AND

MACROMOLECULAR SEQUENCES
Course Code: MIC-403
By: Mahrukh Zakir
Email Address: [email protected]

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 MOLECULAR MARKERS
 Molecular markers are specific sequences or regions of DNA, RNA, or proteins used to identify,
differentiate, or track genetic material in organisms.

 They are widely applied in genetics, evolution, and biotechnology.

 Molecular markers offer numerous advantages, including high precision, broad applicability,
objectivity, and the ability to detect hidden genetic variations. These advantages have made
molecular markers essential in fields such as genetics, medicine, agriculture, and conservation.

 They also provide powerful tools for improving diagnostic accuracy, accelerating breeding
programs, and advancing scientific research.
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 TYPES OF MOLECULAR MARKERS

There are several types of molecular markers, each with distinct

characteristics and applications.
Below are the key types:
 DNA-Based Molecular Markers
 RNA-Based Molecular Markers
 Protein-Based Molecular Markers
 Mitochondrial and Chloroplast Markers
 Epigenetic Markers
 Cytogenetic Markers

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 DNA-BASED MOLECULAR MARKERS

Restriction Fragment Length Polymorphisms (RFLPs)

 Restriction Fragment Length Polymorphisms (RFLPs) are a technique used in
molecular biology to analyze variations in DNA sequences.
 RFLPs occur due to differences in the lengths of restriction enzyme-digested
DNA fragments.
 These differences arise from mutations in DNA that alter restriction enzyme
recognition sites, leading to fragments of varying lengths when the DNA is cut.

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 STEPS

 DNA Extraction and Digestion: DNA is extracted from a sample, and restriction enzymes
(which cut DNA at specific sequences) are used to digest the DNA into fragments.
 Separation by Gel Electrophoresis: The fragments are then separated by size using gel
electrophoresis, which sorts DNA fragments by their length.
 Hybridization with Probes: A labeled DNA probe, which is complementary to a specific
region of the DNA, is used to detect the presence or absence of certain fragments or patterns.
 Analysis: Variations in the fragment sizes between individuals (polymorphisms) are observed,
which can be used to identify genetic differences, paternity, genetic diseases, or evolutionary
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relationships.
RFLPs have been widely used in
genetic mapping, forensic
analysis, and the identification of
genetic markers associated with
diseases.

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 SIMPLE SEQUENCE REPEATS (SSRS) OR
MICROSATELLITES

 Simple Sequence Repeats (SSRs), also known as microsatellites, are short,

repetitive sequences of DNA that consist of a small number of base pairs
repeated in tandem.
 These repeats typically range from 2 to 6 base pairs in length, and the
number of repeats can vary among individuals within a species, making
them highly polymorphic.
 SSRs are widely distributed throughout the genome and can be found in
both coding and non-coding regions.
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 KEY FEATURES OF SSRS
 Repetitive Nature: The core sequence of an SSR is repeated multiple times. For
example, a repeat could be a two-base sequence such as "AG" repeated multiple times
(e.g., AGAGAGAG).
 High Polymorphism: Because the number of repeats can vary greatly between
individuals, SSRs are highly polymorphic and are often used as genetic markers in
research and applications such as genetic mapping and fingerprinting.
 Location: SSRs are found in various regions of the genome, including both coding
(exons) and non-coding (introns, intergenic regions) areas.
 Neutral Markers: Most SSRs are considered neutral markers because their variations
do not typically result in changes to the functioning of the genes, although some can be
located near or within genes that affect phenotype.
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 APPLICATIONS OF SSRS

 Genetic Mapping
 Forensics: SSRs are used in forensic DNA profiling.
 Conservation Biology: SSRs are used to assess genetic diversity in
wildlife populations.
 Breeding Programs: SSR markers are used in plant and animal
breeding to select for desirable traits. For example, SSRs can help
identify genetic markers linked to disease resistance or yield traits in
crops.

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 DETECTION METHODS
 Polymerase Chain Reaction (PCR): PCR is typically used to amplify specific
SSR regions. The amplified fragments are then separated and analyzed by gel
electrophoresis or capillary electrophoresis.
 Fluorescent Labeling: SSR markers can be labeled with fluorescent dyes to
enable detection using automated systems.
 Next-Generation Sequencing (NGS): SSRs can be identified through high-
throughput sequencing, providing a broader and more detailed analysis of the
genetic variation in the entire genome. 10
SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS)

 Single Nucleotide Polymorphisms (SNPs) are the most common

type of genetic variation among individuals.
 A SNP occurs when a single nucleotide (A, T, C, or G) in the DNA
sequence is replaced by a different nucleotide at a specific position
in the genome.
 SNPs can have a significant impact on biological processes, as they
can occur within coding regions (genes) or non-coding regions of
the genome.

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 KEY FEATURES OF SNPS
1. Single Base Pair Change: A SNP involves a change in a single nucleotide, which
could be a substitution of one base pair for another (e.g., A to G, C to T).
2. Common and Widespread: SNPs are abundant in the human genome and occur
approximately once in every 300 nucleotides. There are millions of SNPs across
the genome.
3. Variation: SNPs contribute to genetic diversity within populations. The presence
of different alleles at a given SNP site can influence an individual's traits, health,
and susceptibility to diseases.
4. Intragenic or Intergenic: SNPs can occur within genes (coding regions) or
outside genes (non-coding regions, such as regulatory regions or introns). Their 12

effects may vary depending on their location.

 DETECTION OF SNPS

 Polymerase Chain Reaction (PCR)

 DNA Microarrays
 Next-Generation Sequencing (NGS)
 Sanger Sequencing

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 APPLICATIONS OF SNPS
 Disease Association Studies
 Pharmacogenomics: SNPs play a critical role in understanding how
individuals respond to drugs.
 Personalized Medicine: By identifying SNPs that influence disease risk or
drug response, healthcare providers can tailor medical treatments to
individuals, making them more effective and reducing adverse effects.
 Ancestry and Population Genetics
 Forensic Genetics: paternity testing
 Crop and Livestock Improvement: SNPs are used in agriculture to
identify genes associated with desirable traits in crops (e.g., disease
resistance, yield) and livestock (e.g., growth rate, milk production). 14
AMPLIFIED FRAGMENT LENGTH POLYMORPHISMS
(AFLPS)
 Amplified Fragment Length Polymorphisms (AFLPs) are a
molecular marker technique used to detect genetic variation in a
genome.
 AFLPs combine the principles of restriction enzyme digestion and
selective PCR amplification to generate a large number of markers that
are highly polymorphic, meaning they can identify differences in DNA
sequences between individuals.
 AFLPs are particularly useful in organisms with limited genomic
information or in those that lack prior sequence data.

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 AFLP TECHNIQUE STEPS
1. DNA Extraction: DNA is extracted from the organism of interest.
2. Restriction Enzyme Digestion: The extracted DNA is digested with two different restriction enzymes. The first
enzyme (often a rare-cutter) cuts the DNA at infrequent sites, while the second enzyme (a frequent-cutter) cuts at
more common sequences, resulting in a mixture of DNA fragments of different lengths.
3. Ligation of Adapters: Short synthetic oligonucleotides (adapters) are ligated to the sticky ends of the restriction
fragments. These adapters contain primer binding sites for subsequent amplification.
4. Selective PCR Amplification: The ligated DNA is subjected to PCR amplification using primers that are
complementary to the adapter sequences and a few nucleotides from the restriction site. The amplification is
selective because the primers are designed to amplify only a subset of the fragments, based on a specific sequence at
the restriction enzyme sites. This step enriches for fragments of interest.
5. Gel Electrophoresis: The PCR products are separated by size using gel electrophoresis. The resulting banding
pattern represents the AFLP profile of the individual.
6. Analysis: The bands are scored for the presence or absence of specific fragments. These patterns are used to assess
genetic variation across individuals or populations. 16
APPLICATIONS OF AFLPS
 Genetic Diversity Studies
 Marker-Assisted Selection
 Phylogenetic Analysis
 Conservation Genetics
 Forensic Identification
 Population Genetics
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RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD)

 Random Amplified Polymorphic DNA (RAPD) is a molecular technique

used to detect genetic variation by amplifying random segments of an
organism's genome using short, arbitrary primers.
 RAPD is a type of PCR-based method that does not require prior knowledge
of the sequence of the genome, making it an accessible and versatile tool for
genetic analysis.
 It is often used in biodiversity studies, genetic mapping, and marker-assisted
selection, particularly in species with limited genomic information.
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 RNA-BASED MOLECULAR MARKERS

Expressed Sequence Tags (ESTs)

• ESTs are short DNA sequences that are transcribed from mRNA. These sequences represent
expressed genes in the genome.
• ESTs help identify functional genes and serve as markers for gene expression analysis.
• Applications:
• Functional genomics.
• Identifying differentially expressed genes in disease or stress conditions.

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 PROTEIN-BASED MARKERS

Protein Polymorphisms (Allozymes)

• Allozymes refer to variant forms of enzymes that differ in their amino acid sequence.
These differences can be detected using electrophoresis.
• Allozyme markers are often used to detect genetic variation in populations based on
enzyme activity.
• Applications:
• Population genetics studies.
• Investigating evolutionary relationships.
• Assessing genetic diversity in species.
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 Isoenzymes
 Isoenzymes are different molecular forms of the same enzyme that catalyze the
same reaction but differ in their amino acid sequence.
• Isoenzymes can be used as markers for identifying specific genetic traits or for
tracing parental lineages.
• Applications:
• Species identification.
• Study of genetic diversity in plant and animal populations.

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 EPIGENETIC MARKERS
DNA Methylation Markers
• DNA methylation involves the addition of methyl groups to DNA, which can
affect gene expression without changing the DNA sequence.
• Methylation patterns are important in gene regulation and can be used as
biomarkers for diseases like cancer.
• Applications:
• Disease diagnostics, especially cancer.
• Epigenetic studies and gene regulation.

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 Histone Modification Markers
• Modifications to histone proteins, such as acetylation, methylation, and
phosphorylation, can influence chromatin structure and gene expression.
• These markers help in studying gene regulation and chromatin dynamics.
• Applications:
• Epigenetic research.
• Cancer and developmental disorder studies.

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 MACROMOLECULAR SEQUENCES
 Macromolecular sequences refer to the sequence of monomers (nucleotides for
nucleic acids, amino acids for proteins) that make up larger biological
macromolecules, such as DNA, RNA, and proteins.
DNA Sequences:
 Gene sequences: DNA sequences of genes code for proteins and are crucial for understanding
gene function and regulation. Sequencing technologies allow for the mapping of entire genomes,
enabling researchers to identify specific genes and their mutations.
 Genetic variation: DNA sequences reveal variations within populations, such as mutations,
insertions, deletions, and SNPs. These variations are often used as markers for identifying
genetic diseases or studying evolutionary changes in organisms.
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 PROTEIN SEQUENCES

Amino acid sequences:

 Proteins are made up of chains of amino acids.
 The specific sequence of these amino acids determines the structure and function
of the protein.
Protein domains and motifs:
 Specific sequences within proteins, such as functional motifs or domains, are
important for understanding protein function.
 Molecular markers can be used to identify conserved protein domains across
species, which are often linked to essential biological processes.
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 RNA SEQUENCES
 mRNA sequences: Messenger RNA (mRNA) sequences carry genetic
information from the DNA to the ribosome for protein synthesis.
 Non-coding RNAs: RNA molecules like microRNAs and long non-
coding RNAs play key regulatory roles in gene expression. Changes in
these sequences are used as markers for diseases and regulatory
networks in cells.

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 APPLICATIONS OF MOLECULAR MARKERS IN
MACROMOLECULAR SEQUENCES

 Genetic Mapping and Gene Discovery

 Evolutionary Studies
 Breeding and Agricultural Biotechnology
 Forensic DNA Analysis
 Medical Diagnostics
 Functional Genomics

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