Molecular Marker Types Using Both Restriction Enzyme and PCR Techniques

Which one of the following molecular marker types uses combination of both restriction enzyme and PCR techniques?
(1) SSR
(2) AFLP
(3) SNP
(4) RAPD

The correct answer is (2) AFLP (Amplified Fragment Length Polymorphism).

Introduction to Molecular Markers

Molecular markers are essential tools in genetics and molecular biology for identifying variations in DNA sequences. They help in:

• Genetic mapping
• Marker-assisted selection
• Population genetics
• Phylogenetic analysis

Among the various molecular markers, AFLP (Amplified Fragment Length Polymorphism) stands out because it combines the use of restriction enzymes and PCR amplification to generate a large number of polymorphic markers.

✅ Correct Answer: (2) AFLP

AFLP (Amplified Fragment Length Polymorphism) is a highly sensitive and reproducible method for detecting DNA polymorphisms.

Explanation of Molecular Marker Types

1. SSR (Simple Sequence Repeats) – Incorrect

• SSRs, also known as microsatellites, are short tandem repeats (1–6 bp) repeated multiple times in a DNA sequence.
• SSR analysis involves:
  • Designing primers flanking the repeat region.
  • PCR amplification to detect the length variations of repeats.
• No restriction enzyme is required in SSR analysis.

❌ No restriction enzyme used – Only PCR-based technique.

2. AFLP (Amplified Fragment Length Polymorphism) – Correct

• AFLP is a combination of restriction digestion and PCR amplification.
• Procedure:
  • DNA is digested using restriction enzymes (e.g., EcoRI and MseI).
  • Adapter sequences are ligated to the digested fragments.
  • Selective amplification is performed using PCR primers complementary to adapter sequences.
  • The amplified fragments are separated using gel electrophoresis or capillary electrophoresis.
• AFLP generates a large number of polymorphic markers, providing high resolution for genetic diversity studies.

✅ Uses both restriction enzymes and PCR amplification.

3. SNP (Single Nucleotide Polymorphism) – Incorrect

• SNPs are single-base variations in DNA sequences.
• SNP detection methods include:
  • PCR-based methods (e.g., TaqMan assay, allele-specific PCR).
  • Hybridization-based methods (e.g., DNA microarrays).
  • Sequencing-based methods.
• Restriction enzymes are not typically involved in SNP analysis.

❌ No restriction enzyme required – SNPs are detected using PCR or sequencing methods.

4. RAPD (Random Amplified Polymorphic DNA) – Incorrect

• RAPD is based on the random amplification of DNA segments using short, arbitrary primers.
• Procedure:
  • PCR is performed using a single short primer.
  • Polymorphisms are identified based on differences in amplified fragment size.
• No restriction enzyme is used in RAPD.

❌ No restriction enzyme required – Only PCR-based technique.

How AFLP Works

1. DNA Extraction and Restriction Digestion

• DNA is extracted from the sample.
• Two different restriction enzymes (frequently a rare cutter and a frequent cutter) are used to digest the DNA.

2. Adapter Ligation

• Synthetic adapters are ligated to the sticky ends of the digested fragments.
• These adapters serve as primer-binding sites for selective amplification.

3. Selective PCR Amplification

• PCR amplification is performed using primers complementary to the adapter sequences.
• Primers may include additional selective nucleotides to reduce background noise.

4. Electrophoretic Separation

• Amplified fragments are separated by:
  • Polyacrylamide gel electrophoresis (PAGE)
  • Capillary electrophoresis
• Fragments are detected using:
  • Radioactive labeling
  • Fluorescent dyes

5. Data Analysis

• The size and intensity of the fragments are recorded.
• AFLP profiles are compared to identify polymorphisms and genetic differences.

Advantages of AFLP Technique

✔ High Resolution – Detects small variations in DNA.
✔ High Reproducibility – Results are consistent across different experiments.
✔ No Prior Sequence Information Needed – Can be applied to any organism.
✔ High Polymorphism Detection – AFLP generates numerous polymorphic markers.

Applications of AFLP

1. Genetic Mapping

• AFLP is used for generating high-resolution genetic maps.
• It helps in locating genes linked to specific traits.

2. Population Genetics

• AFLP is widely used to assess genetic diversity in populations.
• It helps in studying gene flow and population structure.

3. Evolutionary Studies

• AFLP provides insight into evolutionary relationships among species.
• It helps in phylogenetic analysis and species differentiation.

4. Plant and Animal Breeding

• AFLP assists in marker-assisted selection (MAS).
• It is used to identify desirable traits for breeding programs.

Comparison of Molecular Marker Techniques

Why AFLP is the Best Choice

• AFLP combines restriction enzyme digestion and PCR amplification, making it suitable for detecting DNA polymorphisms.
• Unlike SSRs and RAPD, AFLP generates a higher number of markers with greater reproducibility and sensitivity.