Skip to main content

AFLP--Amplified Fragment Length Polymorphism






AFLP is a PCR-based DNA fingerprinting technique combining restriction digestion and selective PCR amplification of genomic DNA fragments.
Developed by Vos et al., 1995.
AFLP detects DNA polymorphisms at the genomic level and is highly reproducible and sensitive.
Used in genetic mapping, diversity studies, phylogenetics, and marker-assisted selection.


Principle


AFLP relies on restriction digestion of genomic DNA, followed by ligation of adaptors and PCR amplification of a subset of fragments.
Polymorphism arises due to variations in restriction sites, fragment length, insertions, or deletions.


Key idea:


Restriction digestion → Adaptor ligation → Selective amplification → Gel separation → Detection of polymorphic bands
Materials Required
Genomic DNA
Restriction enzymes (usually EcoRI and MseI)
Adaptors complementary to restriction sites
PCR reagents: Taq polymerase, dNTPs, buffer, Mg²⁺
Primers complementary to adaptors with selective nucleotides
Thermal cycler
Polyacrylamide or agarose gel electrophoresis system
DNA staining dyes or fluorescent labels


Procedure

Step 1: DNA Isolation

Extract high-quality genomic DNA from tissue, blood, or cells.

Step 2: Restriction Digestion

Digest DNA with two restriction enzymes, usually:
EcoRI (rare cutter, 6-base recognition)
MseI (frequent cutter, 4-base recognition)


Step 3: Adaptor Ligation


Ligate synthetic oligonucleotide adaptors to the sticky ends of DNA fragments.
Adaptors serve as primer binding sites for PCR amplification.


Step 4: Pre-selective PCR Amplification


Use primers complementary to adaptors without selective nucleotides.
Amplifies all fragments ligated to adaptors.
Step 5: Selective PCR Amplification
Use primers with 1–3 selective nucleotides at the 3’ end.
Only a subset of fragments is amplified, generating manageable band numbers.


Step 6: Gel Electrophoresis


Separate amplified fragments on polyacrylamide or high-resolution agarose gel.
Visualize polymorphic bands using fluorescent or radioactive labels.


Step 7: Data Analysis


Score presence (1) or absence (0) of bands.
Use for genetic similarity, diversity, mapping, or phylogenetic studies.


Diagram in Words




Genomic DNA → Restriction Enzyme Digestion (EcoRI + MseI)
      ↓
Ligate Adaptors to Sticky Ends
      ↓
Pre-selective PCR (All fragments)
      ↓
Selective PCR (Subset of fragments)
      ↓
Polyacrylamide Gel Electrophoresis
      ↓
Visualization & Scoring of Polymorphic Bands
Characteristics of AFLP
Highly reproducible
High polymorphism detection
Dominant marker (cannot distinguish heterozygotes from homozygotes)
Genome-wide coverage
Requires no prior sequence information.


Merits of AFLP


Detects high number of polymorphisms per assay
Highly reproducible compared to RAPD
No prior sequence information needed
Can be applied to plants, animals, and microbes
Suitable for population genetics, phylogenetics, and genetic mapping.

Limitations of AFLP


Dominant marker: Cannot distinguish heterozygotes
Technically complex: Requires multiple steps (digestion, ligation, two PCRs)
Expensive: Requires restriction enzymes, primers, and fluorescent labels
Band identification is not locus-specific
Less useful for marker-assisted breeding requiring co-dominant markers.


Applications of AFLP


Genetic diversity and population studies
Phylogenetic and evolutionary studies
Construction of genetic maps
Marker-assisted selection (MAS) in breeding programs
DNA fingerprinting
Detection of somaclonal variation in tissue-cultured plants
Microbial strain typing

Conclusion


AFLP is a powerful, highly sensitive, and reproducible DNA fingerprinting technique.
It is suitable for genome-wide polymorphism analysis in species with unknown genomes.
Limitations include dominance, technical complexity, and cost, but its high-throughput capability makes it a widely used molecular marker in genetics, breeding, and phylogenetics.




1. AFLP stands for:
A. Amplified Fragment Length Polymorphism
B. Arbitrary Fragment Length Polymorphism
C. Amplified Fluorescent Labeled Polymorphism
D. Allele Fragment Length Polymorphism
Answer: A

2. AFLP combines which two techniques?
A. RAPD + RFLP
B. RFLP + SSR
C. PCR + Restriction digestion
D. Southern blot + PCR
Answer: C

3. AFLP was developed in:
A. 1985
B. 1990
C. 1995
D. 2000
Answer: C


4. AFLP is a:
A. Protein marker
B. Dominant DNA marker
C. Co-dominant DNA marker
D. Morphological marker
Answer: B


5. AFLP requires prior DNA sequence information:
A. Yes
B. No
Answer: B
Principle and Procedure
6. AFLP detects polymorphism due to:
A. Restriction site variations
B. Fragment length differences
C. Insertions or deletions
D. All of the above
Answer: D
7. AFLP uses which restriction enzymes commonly?
A. EcoRI and MseI
B. HindIII and BamHI
C. TaqI and PstI
D. None of the above
Answer: A
8. Adaptors are ligated to DNA fragments in AFLP to:
A. Digest DNA
B. Provide primer binding sites
C. Label DNA
D. Visualize DNA
Answer: B
9. AFLP involves how many PCR steps?
A. One
B. Two (pre-selective and selective)
C. Three
D. Four
Answer: B
10. Selective PCR primers in AFLP have:
A. No additional nucleotides
B. 1–3 selective nucleotides at 3’ end
C. 10 extra nucleotides at 5’ end
D. Random length nucleotides
Answer: B
11. AFLP fragments are separated by:
A. Agarose or polyacrylamide gel electrophoresis
B. SDS-PAGE
C. Native PAGE only
D. Capillary electrophoresis
Answer: A
12. Visualization of AFLP bands is commonly done using:
A. Fluorescent dyes
B. Radioactive labeling
C. Silver staining
D. Any of the above
Answer: D
13. AFLP polymorphism is scored as:
A. Homozygote/heterozygote
B. Presence (1) or absence (0)
C. Protein intensity
D. RNA expression
Answer: B
14. AFLP requires DNA of:
A. Very large amount
B. Moderate amount
C. Very small amount
D. RNA contamination
Answer: B
15. AFLP is considered more reproducible than:
A. RAPD
B. SSR
C. RFLP
D. Morphological markers
Answer: A
Advantages
16. AFLP can detect:
A. Hundreds of polymorphic fragments per assay
B. Only a few fragments
C. Single-locus polymorphism only
D. Only protein variation
Answer: A
17. AFLP does not require:
A. Restriction digestion
B. Southern blotting
C. PCR amplification
D. Primers
Answer: B
18. AFLP is suitable for:
A. Plants
B. Animals
C. Microorganisms
D. All of the above
Answer: D
19. AFLP is useful for:
A. Population genetics
B. Phylogenetic studies
C. Marker-assisted selection
D. All of the above
Answer: D
20. AFLP is advantageous because it:
A. Requires no prior genome information
B. Is highly reproducible
C. Detects genome-wide polymorphism
D. All of the above
Answer: D
Limitations
21. AFLP is a:
A. Co-dominant marker
B. Dominant marker
Answer: B
22. AFLP cannot distinguish:
A. Polymorphic fragments
B. Homozygotes from heterozygotes
C. Genetic diversity
D. Band pattern differences
Answer: B
23. AFLP requires:
A. Restriction enzymes, ligation, and two PCRs
B. Only PCR
C. Only Southern blotting
D. Only primers
Answer: A
24. AFLP is:
A. Simple and cheap
B. Technically complex and expensive
C. Dominant and locus-specific
D. Co-dominant and fast
Answer: B
25. AFLP fragments are generally:
A. Locus-specific
B. Randomly distributed
Answer: B
Applications
26. AFLP is widely used for:
A. Genetic mapping
B. Phylogenetics
C. Population studies
D. All of the above
Answer: D
27. AFLP is used in plants for:
A. Variety identification
B. Disease resistance mapping
C. Hybrid verification
D. All of the above
Answer: D
28. AFLP can detect:
A. Somaclonal variation
B. DNA fingerprinting
C. Genome-wide polymorphisms
D. All of the above
Answer: D
29. AFLP is suitable for species with:
A. Unknown genome sequences
B. Fully sequenced genomes only
Answer: A
30. AFLP has largely replaced RAPD for:
A. Low-resolution studies
B. High-resolution fingerprinting
Answer: B
Technical Knowledge
31. AFLP pre-selective PCR uses:
A. Primers complementary to adaptors only
B. Primers with selective nucleotides
Answer: A
32. Selective PCR uses primers with:
A. No selective nucleotides
B. 1–3 selective nucleotides
Answer: B
33. AFLP is sensitive to:
A. PCR conditions
B. DNA quality
C. Enzyme activity
D. All of the above
Answer: D
34. AFLP is a:
A. Hybridization-based technique
B. PCR-based technique
Answer: B
35. AFLP produces:
A. Few bands
B. Hundreds of bands per assay
Answer: B
Comparison with Other Markers
36. Compared to RAPD, AFLP is:
A. Less reproducible
B. More reproducible
Answer: B
37. Compared to RFLP, AFLP:
A. Is PCR-based and faster
B. Requires Southern blot
Answer: A
38. Compared to SSR, AFLP:
A. Requires prior sequence info
B. Does not require prior sequence info
Answer: B
39. AFLP is dominant, whereas SSR is:
A. Dominant
B. Co-dominant
Answer: B
40. AFLP is preferred for:
A. High-resolution genome-wide studies
B. Low-resolution studies
Answer: A
Advanced Applications
41. AFLP is useful in:
A. Plant breeding programs
B. Animal population studies
C. Microbial strain typing
D. All of the above
Answer: D
42. AFLP data is usually analyzed using:
A. Binary scoring of bands
B. Protein gels
C. RNA profiling
Answer: A
43. AFLP is highly suitable for:
A. Phylogenetic studies of closely related species
B. Morphological analysis
Answer: A
44. AFLP allows:
A. Detection of large and small fragment polymorphisms
B. Detection of only large fragments
Answer: A
45. AFLP is widely used in:
A. Forensic science
B. Conservation biology
C. Genetic mapping
D. All of the above
Answer: D
Miscellaneous
46. AFLP requires:
A. Restriction digestion
B. Adaptor ligation
C. Two PCR steps
D. All of the above
Answer: D
47. AFLP primers are designed:
A. Randomly complementary to adaptors
B. From coding regions only
Answer: A
48. AFLP polymorphism arises due to:
A. Variation in restriction sites
B. Sequence insertions/deletions
C. Mutations at primer binding sites
D. All of the above
Answer: D
49. AFLP produces:
A. Reproducible fingerprints
B. Irreproducible patterns
Answer: A
50. AFLP is considered more advanced than RAPD because:
A. Higher reproducibility
B. Higher resolution
C. Genome-wide coverage
D. All of the above
Answer: D

Labels:

Comments

Post a Comment

Popular Posts

Genetically modified microbes - biodegradation, biopesticides, bioremediation, mineral leaching and biofertilizers.

 Genetically Modified Microbes (GMMs) covering biodegradation, biopesticides, bioremediation, mineral leaching and biofertilizers.  Genetically Modified Microbes (GMMs) Introduction Genetically Modified Microbes (GMMs) are microorganisms such as bacteria, fungi, yeast or algae whose genetic material has been altered using recombinant DNA technology to enhance or introduce desirable traits. These microbes are engineered to improve efficiency, specificity and speed of biological processes useful in agriculture, industry and environmental management. GMMs play a vital role in sustainable development by reducing dependence on chemical fertilizers, pesticides and polluting industrial processes. 1. Genetically Modified Microbes in Biodegradation Definition Biodegradation is the microbial breakdown of complex organic pollutants into simpler, non-toxic substances. Role of GMMs Natural microbes often degrade pollutants slowly. Genetic modification enhances: Enzyme activity Substrate sp...
Post a Comment
Read more

Mapping of DNA

DNA MAPPING   1. Introduction DNA mapping refers to the process of determining the relative positions of genes or DNA sequences on a chromosome. It provides information about the organization, structure, and distance between genetic markers in a genome. DNA mapping is an essential step toward genome sequencing, gene identification, disease diagnosis, and genetic engineering. DNA maps serve as roadmaps that guide researchers to locate specific genes associated with traits or diseases. 2. Objectives of DNA Mapping To locate genes on chromosomes To determine the order of genes To estimate distances between genes or markers To study genome organization To assist in genome sequencing projects. 3. Principles of DNA Mapping DNA mapping is based on: Recombination frequency Physical distance between DNA fragments Hybridization of complementary DNA Restriction enzyme digestion Use of genetic markers The closer two genes are, the less frequently they recombine during meiosis. 4 . Types of DNA...
2 comments
Read more

protoplast fusion

Protoplast Fusion – Detailed Notes 1. Definition Protoplast fusion is a technique in which two or more protoplasts (cells without cell walls) are fused to form a single hybrid cell. It is widely used in plant biotechnology for hybridization, gene transfer, and somatic hybrid production. Also called somatic hybridization or somatic cell fusion. 2. Principle Cell wall removal: Plant cells are treated with cell wall-degrading enzymes (cellulase, pectinase) to generate protoplasts. Fusion of protoplasts: The naked cells are induced to fuse physically or chemically. Hybrid cell formation: Nuclei from different protoplasts combine to form a heterokaryon. Regeneration: The hybrid cell regenerates a new cell wall and divides, eventually forming a somatic hybrid plant. Key Concept: Protoplast fusion bypasses sexual incompatibility barriers, allowing hybridization between distant species or genera. 3. Steps in Protoplast Fusion Step 1: Isolation of Protoplasts Plant tissues (leaves, callus, cell...
Post a Comment
Read more

✩‧₊ Plaque Blotting Technique

Plaque Blotting Technique *ੈ✩‧₊˚༺☆༻*ੈ✩‧₊˚*ੈ✩‧₊˚༺☆༻*ੈ✩‧₊˚ Introduction Plaque blotting is a molecular biology screening technique used to identify specific DNA or RNA sequences present in bacteriophage plaques formed on a bacterial lawn. It is especially useful in the screening of recombinant phage libraries such as λ (lambda) phage genomic or cDNA libraries. This technique combines: Plaque assay (to isolate individual phage clones) Blotting technique (to transfer nucleic acids onto a membrane) Hybridization (to detect specific sequences using labeled probes) Principle of Plaque Blotting The principle of plaque blotting is based on nucleic acid hybridization. Each plaque represents a clone of phage particles containing identical DNA. DNA from phage particles in plaques is: Released Denatured into single strands Transferred onto a nitrocellulose or nylon membrane The membrane is incubated with a labeled DNA/RNA probe complementary to the target sequence. Hybridization between probe and t...
Post a Comment
Read more

Protein Sequence DatabasesPIR, SWISS-PROT and TREMBEL

Protein Sequence Databases PIR, SWISS-PROT and TREMBEL 1. Introduction Protein sequence databases are biological databases that store information about amino acid sequences of proteins, along with their functional, structural, and biochemical characteristics. Since proteins are the functional molecules of the cell, protein databases are essential for understanding gene expression, metabolism, enzymatic activity, signaling pathways, and evolution. Protein sequence databases mainly contain data derived from translated nucleotide sequences and experimental protein studies. 2. Types of Protein Sequence Databases Protein sequence databases are broadly classified into: A. Primary Protein Databases Contain original protein sequence data Minimal or no manual annotation B. Secondary Protein Databases Derived from primary databases Provide curated functional and structural information C. Composite Protein Databases Combine protein data from multiple sources Reduce redundancy 3. Protein Informati...
Post a Comment
Read more

Secondary Databases (PROSITE, PRINTS, BLOCKS)

Secondary Databases (PROSITE, PRINTS, BLOCKS  Secondary Databases Introduction Biological databases are broadly classified into primary and secondary databases. Primary databases store raw experimental data (e.g., nucleotide or protein sequences), whereas secondary databases contain derived information obtained by analyzing primary sequence data. Secondary databases are mainly used to: Identify protein families Detect conserved motifs, patterns, and domains Predict protein function Study structure–function relationships Examples of secondary databases include PROSITE, PRINTS, BLOCKS, Pfam, etc. 1. PROSITE Database Definition PROSITE is a secondary database that documents protein domains, families, and functional sites in the form of patterns and profiles. Developed by Swiss Institute of Bioinformatics (SIB) Maintained along with UniProt Principle PROSITE is based on the idea that functionally important regions of proteins are conserved during evolution. These conserved regions can ...
Post a Comment
Read more

Protein Structure Database (PDB)

Protein Structure Database (PDB) Introduction The Protein Structure Database (PDB) is the primary global repository for the three-dimensional (3D) structures of biological macromolecules such as proteins, nucleic acids, and protein–ligand complexes. These structures are determined experimentally using techniques like X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, and Cryo-Electron Microscopy (Cryo-EM). PDB plays a vital role in understanding: Protein structure and function Molecular interactions Drug discovery and design Structural biology and bioinformatics History and Development Established in 1971 Founded by Brookhaven National Laboratory (USA) Initially contained only 7 protein structures Now maintained by the Worldwide Protein Data Bank (wwPDB) Members of wwPDB RCSB PDB (USA) PDBe (Europe) PDBj (Japan) BMRB (Biological Magnetic Resonance Data Bank) Objectives of PDB To collect, store, and distribute 3D structural data of biomolecules To provide free and ope...
Post a Comment
Read more

♡Introduction to Bioinformatics: Definition and History of Bioinformatics Internet. Computational Biology and Bioinformatics

Introduction to Bioinformatics: Definition and History of Bioinformatics Internet.  Computational Biology and Bioinformatics ﮩ٨ـﮩﮩ٨ـ♡ﮩ٨ـﮩﮩ٨ـﮩ٨ـﮩﮩ٨ـ♡ﮩ٨ـﮩﮩ٨ﮩ٨ـﮩﮩ٨ـ♡ﮩ٨ـﮩ Definition of Bioinformatics Bioinformatics is an interdisciplinary field that combines biology, computer science, mathematics, and statistics to collect, store, analyze, and interpret large volumes of biological data. It mainly deals with molecular biology data such as DNA, RNA, protein sequences, gene expression data, and biological networks. Bioinformatics helps in understanding biological processes at the molecular level using computational tools. It plays a crucial role in modern biological research, especially after the availability of whole genome sequences. According to NIH, “Bioinformatics is the application of computational tools to capture and interpret biological data.” History and Evolution of Bioinformatics Early Beginnings (Pre-1970) The roots of bioinformatics date back to the 1950s–1960s. In 1953, Wat...
Post a Comment
Read more

SCAR (Sequence Characterized Amplified Region) Markers

SCAR (Sequence Characterized Amplified Region) Markers   Introduction SCAR markers are PCR-based DNA markers derived from RAPD, AFLP, or other random markers. Developed by Paran and Michelmore in 1993 to convert dominant, less reproducible markers into specific, reproducible, co-dominant markers. SCAR markers are locus-specific, reproducible, and sequence-characterized, making them ideal for marker-assisted selection (MAS). Principle SCAR markers are designed based on known DNA sequences obtained from cloned RAPD/AFLP fragments. Specific primers (18–24 bp) are synthesized to amplify a single, defined locus. The PCR amplification of this region generates a distinct band, which is highly reproducible and can distinguish homozygotes from heterozygotes if designed as co-dominant. Key idea: Random marker (e.g., RAPD) → Cloning & sequencing → Design specific primers → PCR → SCAR marker Materials Required Genomic DNA from the organism Specific primers (18–24 bp) designed from sequence...
Post a Comment
Read more

Fourth Semester M.Sc. Degree Examination, June 2022BotanySpecial Paper II - Elective BO 242 a BIOTECHNOLOGY

N-6278 Fourth Semester M.Sc. Degree Examination, June 2022 Botany Special Paper II - Elective BO 242 a BIOTECHNOLOGY - (2013-2018 Admission) Time: 3 Hours Max. Marks 75 Give Illustrations wherever necessary. 1. Answer the following questions. 1. Define a calliclone. 2. Which fungi helps in fermentation? 3. How is hairy root culture induced in plants? 4. Name a microbe that produces organic acid. 5. What is the advantage of chromosome elimination technique? 6. Describe the significance of PIPS. 7. What is elicitation in plant tissue culture? 8. Describe the term bioconversion. 9. Which is the most successful chemical used for protoplast fusion? 10. Define cybridization. (10x1 =10 Marks) II. Answer the following questions in not more than 50 words . 11. (a) Give an account on signature tagged mutagenesis. OR (b) Write notes on cytoplasts and their importance? 12. (a) Give an account of gene theft. OR (b) Describe induced androgenesis and gynogenesis for haploid plant production. 13. (a) ...
Post a Comment
Read more