Cellular & Molecular Pharmacology: Gene Mapping .pptx

Gene mapping is the process of identifying the location of genes on a chromosome and determining the distance between them. It helps in understanding genetic disorders, inheritance patterns, and genome function. Two types of gene mapping that are Linkage mapping and Physical mapping.

1. GENE MAPPING Presented by: Vaishnavi J. Aware Guided by: Dr. Pavankumar Wankhede Department of Pharmacology Dr. D. Y. Patil College of Pharmacy, Akurdi , Pune 411044

2. Definition Gene mapping is the process of determining the location (loci or locus) of gene and distance between gene on a chromosome It involves identifying the specific position of a gene or a group of genes within the genome and understanding their relationship to other genes, traits or disease. This process can be done using techniques such as linkage analysis, genetic markers, and various molecular biology methods. Gene mapping is essential for understanding genetic inheritance, discovering disease-causing gene, and developing treatments for genetic disorders.

3. Types of gene mapping There are two types of gene mapping: Genetic mapping or linkage mapping Physical mapping

4. Genetic mapping It is also known as linkage mapping or chromosome mapping. In linkage mapping genetic technique is use to construct maps showing the position of gene. Linkage mapping determines the corresponding location between genes on chromosome which are likely to be inherited together during meiosis, a process known as genetic linkage. The distance between genes is measured in centimorgans ( cM ), where 1 cM corresponds to a 1% chance of recombination between markers. DNA markers are used for genetic mapping : Restriction Fragment Length Polymorphism (RFLP) Simple Sequence Repeats (SSRs) or Microsatellites Single Nucleotide Polymorphisms (SNPs) Amplified Fragment Length Polymorphisms (AFLPs) Random Amplified Polymorphic DNA (RAPD) Cleaved Amplified Polymorphic Sequences (CAPS) Sequence-Tagged Sites (STS) Expressed Sequence Tags (ESTs)

5. Restriction Fragment Length Polymorphisms (RFLPs) RFLP is a molecular technique used to detect variations in the DNA sequence of individuals. It was one of the first DNA-based markers used in genetic mapping. RFLPs are based on the presence or absence of specific  restriction enzyme recognition sites  in the genome, leading to differences in the length of DNA fragments after digestion. Caused by mutations at restriction enzyme recognition sites (e.g., insertions, deletions, or point mutations) that result in DNA fragments of different lengths. Detected using Southern blotting, where DNA is digested, separated by gel electrophoresis, and hybridized with a labeled DNA probe. Used in early genetic mapping studies, plant and animal breeding, and evolutionary studies.

6. Simple Sequence Repeats ( SSRs ) / Microsatellites Repeated short DNA sequences (e.g., (CA)ₙ, (GT)ₙ) found throughout the genome. The number of repeats varies between individuals. Variations in the number of repeat units caused by DNA replication slippage. Detected using PCR with primers flanking the repeat region, followed by gel electrophoresis or capillary electrophoresis. Widely used in plant and animal breeding, forensics, population genetics, and human genetic mapping.

7. Single Nucleotide Polymorphisms (SNPs) Single-base pair changes in the DNA sequence (e.g., A→G or C→T) at specific loci. Point mutations that become fixed in a population or species. Detected using SNP genotyping arrays, PCR, DNA sequencing, or high-throughput techniques like next-generation sequencing (NGS). SNPs are widely used in human disease studies (GWAS), population genetics, and personalized medicine.

8. Amplified Fragment Length Polymorphisms (AFLPs) Polymorphisms in DNA fragment lengths produced by selective PCR amplification of digested genomic DNA. Variations in restriction enzyme recognition sites or the regions flanking them. Detected using polyacrylamide gel electrophoresis or capillary electrophoresis after PCR amplification. Used in plant and animal breeding, population genetics, and evolutionary studies.

9. Random Amplified Polymorphic DNA (RAPD) Polymorphisms generated by amplifying random DNA sequences using short arbitrary primers in PCR. Variations in primer-binding sites due to insertions, deletions, or mutations. Detected by separating PCR products on agarose gels and visualizing banding patterns. Used for genetic diversity studies, species identification, and quick screening of genetic variation.

10. Cleaved Amplified Polymorphic Sequences (CAPS) Polymorphisms detected by digesting PCR products with restriction enzymes. Mutations that create or destroy restriction sites within amplified regions. Detected by gel electrophoresis of restriction enzyme-digested PCR products. Used for mapping genes associated with specific traits or diseases, especially in plants.

11. Sequence-Tagged Sites (STS) Short, unique, and known sequences of DNA that serve as reference points in the genome. Naturally occurring, unique regions of DNA. Detected by PCR amplification of known regions using specific primers. Used in physical mapping, whole-genome sequencing, and large genome projects like the Human Genome Project.

12. Expressed Sequence Tags (ESTs) Short sub-sequences of cDNA (complementary DNA) corresponding to expressed genes. Derived from mRNA sequences, representing the transcribed regions of the genome. Detected via PCR amplification and sequencing of cDNA libraries. Used to identify expressed genes, develop gene-based markers, and study gene expression.

13. Physical mapping Physical mapping is a method used to determine the physical arrangement of DNA sequences within a genome. Unlike genetic mapping, which relies on the frequency of recombination to estimate distances between markers, physical mapping determines the actual distances between DNA fragments, often measured in base pairs (bp). Physical maps provide much higher resolution than genetic maps. They reveal the precise location of DNA sequences, including genes, regulatory regions and repetitive elements. The techniques used in physical mapping: Restriction mapping Fluorescence In Situ Hybridization (FISH) Clone-based mapping Optical mapping Sequence-tagged Sites (STSs) Radiation Hybrid mapping

14. Restriction Mapping Involves determining the positions of restriction enzyme sites on a DNA molecule. Provides a map showing the relative locations of these sites along the DNA. Process : Digest the DNA with restriction enzymes (e.g., EcoRI , HindIII ). Separate the resulting fragments by gel electrophoresis. Compare fragment patterns from single, double, and multiple enzyme digestions. Reconstruct the positions of the enzyme recognition sites on the DNA. Used to map small DNA molecules like plasmids. Provides a framework for further sequencing.

15. Fluorescence In Situ Hybridization (FISH) A technique that uses fluorescently labeled probes to detect specific DNA sequences on chromosomes. Process : Prepare a chromosome spread on a microscope slide. Denature the DNA to make it single-stranded. Hybridize fluorescently labeled probes complementary to the target sequence. Visualize the probes under a fluorescence microscope. Localization of specific genes or markers. Detection of chromosomal abnormalities. Comparative genomics and evolutionary studies.

16. Clone-based Mapping Involves creating overlapping clones of large DNA fragments to build a continuous sequence map, known as a contig. Process : Fragment genomic DNA using restriction enzymes or mechanical shearing. Insert DNA fragments into vectors like BACs (Bacterial Artificial Chromosomes) or YACs (Yeast Artificial Chromosomes) . Identify overlaps between clones using hybridization, sequence-tagged sites (STS), or restriction mapping. Assemble clones into a contig. Foundation for genome sequencing projects. Mapping of complex regions in large genomes.

17. Optical Mapping A high-resolution, single-molecule technique to create a map of DNA fragment patterns based on restriction enzyme cleavage or other DNA-breaking methods. Process : Stretch individual DNA molecules on a surface. Treat with restriction enzymes or other DNA-break mechanisms. Visualize the pattern of fragment lengths using microscopy. Scaffolding for genome assembly. Detection of large structural variations.

18. Sequence-Tagged Sites (STS) Mapping Uses unique, short DNA sequences (200-500 bp) as landmarks for mapping. Process : Select STS markers based on known DNA sequences. Amplify markers using PCR from overlapping clones or genomic DNA. Align clones based on shared STS markers. High-resolution mapping of specific genomic regions. Integration with clone-based mapping.

19. Radiation Hybrid Mapping Combines physical and genetic mapping by using radiation-induced chromosome breakage to determine marker order and distances. Process : Expose cells to ionizing radiation to fragment chromosomes. Fuse fragmented human chromosomes with rodent cells to form hybrids. Identify retained human DNA fragments using PCR or hybridization. Analyze the co-retention patterns of markers to deduce their order and distances. Mapping of difficult genomic regions. Integration with genetic maps.

20. Genetic mapping Physical mapping Determines the relative positions of genes/markers based on recombination frequencies during meiosis. 1. Determines the actual physical locations and distances between genes/markers on a DNA molecule. 2. Recombination events during meiosis. 2. D NA sequence or structure (measured in base pairs). 3. Unit of measurement is Centimorgans ( cM ). 3. Unit of measurement is base pairs (bp), kilobases (kb), or megabases (Mb). 4. A linkage map showing relative positions of markers. 4. A physical map showing exact distances between markers. 5. Genetic markers such as SNPs, RFLPs, microsatellites. 5. Physical landmarks such as restriction sites, STS, and cloned DNA fragments. 6. They have lower resolution. 6. They have higher resolution. 7. Applications - Identifying linked traits or genes. - Studying inheritance patterns. - Assisting in breeding programs. 7. Applications - Genome sequencing and assembly. - Locating disease-causing mutations. - Structural variation studies. Difference between Genetic mapping and Physical mapping

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