mol.markers ppt

Introduction

  • Molecular markers are crucial tools in genetic studies and applications.

  • Presenters: Preshita Sanjay Dave (1RV17BT034) and Swathi Giridhar (1RV17BT054).

Molecular Markers

  • Definition: A DNA sequence that is easily detected and monitored for inheritance.

  • Characteristics:

    • Specific DNA fragments identifiable within the whole genome.

    • Found at designated locations in the genome.

    • Used for flagging particular genes or desired characteristics.

Classification of Biological Markers

  • Types:

    • Biochemical: Isozymes, protein binding patterns.

    • Morphological: Based on observed physical traits.

    • Molecular: Based on hybridization methods and PCR techniques.

Molecular Markers Based on PCR

  • Examples include:

    • AP-PCR

    • SCoT

    • RAPD

    • EST

    • ISSR

    • SSR

    • STS

    • AFLP

    • SCAR

RAPD: Random Amplified Polymorphic DNA

  • Technique Overview:

    • PCR-based method using single short oligonucleotide primers randomly selected to amplify DNA fragments across the genome.

Steps in RAPD Analysis

  • Process:

    1. Isolation of DNA

    2. Conduct PCR using a thermocycler

    3. Separate DNA strands

    4. Synthesize complementary strands

    5. Anneal primers

    6. Separate by gel electrophoresis

    7. Detect bands using EtBr staining.

Advantages and Disadvantages of RAPD Analysis

  • Advantages:

    • No prior DNA probes or sequence info needed for primer design.

    • Quick, simple, no blotting or hybridization steps.

    • Low quantity of DNA needed per reaction; can be automated.

    • Low assay costs compared to other marker technologies.

  • Disadvantages:

    • Dominance makes it hard to distinguish between heterozygous and homozygous loci.

    • Quality of PCR product may vary based on many factors: DNA quality, primer mismatches, etc.

    • Rare detection of codominant markers; reproducibility issues.

AP-PCR: Arbitrarily Primed Polymerase Chain Reaction

  • Overview: Modification of RAPD using a single primer (10-15 nucleotides).

  • Technique Variations: Low stringency in initial cycles increases to high stringency later.

Applications of RAPD

  • Used for analysis of genetic diversity across organisms including animals, plants, and microbes.

  • Applications in genome mapping, hybrid purity analysis.

SCoT: Start Codon Targeted Polymorphism

  • Definition: PCR-based technique developed in 2009 for analyzing genetic diversity.

  • Process: Uses 18-mer primers with annealing at 50°C, and standard agarose gel electrophoresis.

Advantages of SCoT

  • Technically simple, highly reproducible with longer primers, and high polymorphism.

  • Can be applied directly to marker-assisted breeding programs.

Disadvantages of SCoT

  • Low reproducibility; PCR conditions significantly impact results.

  • Highly sensitive to changes in conditions.

EST: Expressed Sequence Tags

  • Definition: Short DNA fragments (100-800 nt) representing expressed genes in various tissues.

  • Process of Preparation:

    1. Isolate mRNA from tissue

    2. Reverse transcription to cDNA

    3. Separation of cDNA by electrophoresis

    4. Sequence both ends for comparisons.

Applications of EST

  • Identifies and maps unknown genes, provides insights on gene expression and regulation, and constructs genomic maps.

SSR: Simple Sequence Repeats

  • Definition: Short tandem repeats of 1-6 bases found in eukaryotes, also known as Microsatellites.

  • Types: Based upon their location and the size of the repeat motifs.

Causes of SSR Variation

  • Primarily caused by replication slippage, mutations, and recombination.

SSR Markers

  • Illustrates variation in SSR length using PCR-based methods with specific primers.

Advantages of SSR Markers

  • High co-dominance, hyper variable, unaffected by environmental factors, used in diversity assessments.

  • Applications in genetic diversity, phylogeny, linkage and QTL studies.

ISSR: Inter Simple Sequence Repeats

  • Overview: Uses primers from SSR regions to amplify DNA between two SSR repeats.

Pros and Cons of ISSR

  • Advantages:

    • No need for sequence information, environmentally stable, efficient, needs less DNA.

  • Disadvantages: Low reproducibility, can be locus non-specific.

STS: Sequence Tagged Sites

  • Definition: Short, unique DNA sequences (100-500 bp) recognized once in the genome.

  • Advantages: Useful for quick mapping and data sharing across labs.

  • Disadvantages: Development of new STS can be labor-intensive and requires expertise.

AFLP: Amplified Fragment Length Polymorphism

  • Definition: Leverages SNPs or INDELs that influence restriction site recognition.

  • Process: Involves genomic DNA digestion, ligation with adaptors, and selective PCR amplification.

Advantages of AFLP

  • High sensitivity, reproducibility, and usage in gene library screening.

  • Efficient at revealing genetic diversity.

SCAR: Sequence Characterized Amplified Regions

  • Definition: Derived from RAPD markers but more specific and reproducible.

  • Application: Identify polymorphisms, advantageous for complex traits.

Applications of SCAR Markers

  • Authentication of medicinal herbs, strain selection, and genetic characterization in plants.

RAPD vs. SCAR Comparison

  • RAPD: Less specific, multiple loci, higher variability.

  • SCAR: Highly specific, typically single locus, greater reliability.

Molecular Markers Based on Hybridization

  • Includes techniques like RFLP, MINISATELLITE, and MICROSATELLITE.

RFLP: Restriction Fragment Length Polymorphism

  • Definition: Exploits variations in DNA sequences to distinguish populations/individuals.

  • Steps: DNA isolation, restriction, electrophoresis, blotting, and visualization using probes.

Advantages and Disadvantages of RFLP

  • Advantages: Efficient in genetic mapping, disease determination, and DNA sample source confirmation.

  • Disadvantages: Time-consuming, requires large DNA samples, and labor-intensive isolation processes.

MINISATELLITE

  • Definition: Arrays of tandem repeats (9-100 bp), commonly found in euchromatin.

  • Characteristics: VNTRs allow forensic differentiation.

MICROSATELLITE

  • Characteristics: Polymorphic loci with repeating units of 2-6 bp, useful for constructing DNA profiles.

Biochemical Markers

  • Isozymes: Enzymatic variants, usually co-dominant, important for metabolic activity control.

Methodology for Isozymes

  • Involves protein extraction, electrophoresis, and enzyme detection.

Advantages and Disadvantages of Isozymes

  • Advantages: Rapid identification of outcrosses, cost-effective.

  • Disadvantages: Limited to enzymes detectable in situ.

ALLOZYMES

  • Description: Variants of the same enzyme coded by different allele combinations, good markers for population genetics.

Telomerase as a Molecular Marker

  • Functions: Protects chromosome ends; highly active in cancer cells.

  • Significance: Key target in anticancer therapies.

FISH-DNA Amplification Markers

  • Definition: Detects specific DNA sequences in chromosomes using fluorescent probes.

  • Applications: Used for identifying chromosomal abnormalities in laboratories.

Types of Mapping

  • Genetic Mapping: Uses genetic techniques for map construction based on gene positions.

  • Physical Mapping: Examines DNA molecules for the physical arrangement of sequence features on chromosomes.

Principles of Map-based Cloning

  • Steps to identify tightly linked markers, and to create genomic maps with candidate gene isolation.

Applications of Molecular Markers in Breeding

  • Applications:

    1. Marker-assisted selection for improved efficiency.

    2. Genetic diversity assessments to ensure breeding success.

    3. Genetic mapping for positional information.

    4. Trait manipulation and authentication techniques.

    5. Phylogeny studies for taxonomic classification.