GENE MAPPING AND CLONING OF DISEASE GENE by Paragmoni Bora

This document provides an overview of gene mapping and cloning of disease-related genes. It explains both genetic mapping and physical mapping methods used to determine the locations of genes on chromosomes. Genetic (linkage) mapping relies on polymorphic DNA markers such as RFLPs, SSLPs, and SNPs to identify gene positions based on inheritance patterns. The process of cloning a disease gene involves creating a recombinant DNA molecule containing the target gene, replicating it within host cells, and producing multiple identical gene clones. Techniques like PCR and other molecular tools play a crucial role in identifying, isolating and analyzing disease-associated genes at the DNA level.

1. Presented by Paragmoni Bora Department of pharmacology GENE MAPPING AND CLONING OF DISEASE GENE NETES Institute of Pharmaceutical Science(NIPS), Mirza Guwahati

2. CONTENTS 1. Introduction 5. RFLP(Restriction Fragment length polymorphism ) 2. Types of Mapping 6. Simple Sequence Length Polymorphism (SSLP) 11. Importance of Mapping 8. Demerits 3. Genetic Mapping 7. Single Nucleotide Polymorphism (SNP) 9. Physical Mapping 16. References 4. DNA Markers for Genetic Mapping 10. Genetic Map vs Physical Map 12. Cloning of a Disease Gene 13. Applications of Disease Gene Cloning 14. Basic Steps of Gene Cloning 15. PCR

3. 1. Introduction Gene: A gene is a unit of heredity that is passed from parent to offspring and determines specific characteristics or traits in the organism. It was the first genetic marker to be identified and used in heredity studies. Mapping: Mapping refers to the process of determining the precise location of genes or other genetic elements within a genome relative to identifiable landmarks or reference points.

4. 2. Types of Mapping Genetic mapping: linear description of DNA markers/genes on a given chromosome with closely placed markers being inherited together more often. Physical mapping: physical location on the chromosome, relating more towards exact positioning of gene elements. Comparative mapping: Comparative mapping is the process of comparing the genetic maps of different species to find similarities and differences in their genome organization Genome/genetic maps: graphic representation of the relative positions of genes and DNA sequences.

5. Types of Mapping Genetic Mapping 1 2 Physical Mapping 3 Comparative Mapping Gene sequences. Databases: DNA chips. Linkage Mapping Pedigree Polymorphic markers Cytogenetic mapping. Somatic cell mapping. Radiation hybrid mapping Restriction mapping: PFGE, BAC contigs, sequencing. This Photo by Unknown Author is licensed under CC BY

6. 3. Genetic Mapping Shows the positions of genes or markers on a chromosome. Needs informative markers (that show variation/polymorphism). Requires a population with known relationships (like a family or cross). Works best when markers are close together on the chromosome. Unit of distance: centiMorgan ( cM ). 1 cM = 1% chance of recombination between two markers.

7. 4. DNA Markers for Genetic Mapping Genes are useful markers but not ideal. Mapped features that are not genes are called DNA markers. DNA markers must have at least two alleles to be useful. DNA sequence features that satisfy this requirement are: Restriction fragment length polymorphism (RFLP). Simple sequence length polymorphism (SSLP). Single nucleotide polymorphism (SNP). 1 2 3

8. 5. RFLP ( Restriction fragment length polymorphism ) RFLP is the first type of DNA marker to be studied. Restriction enzymes cut DNA at specific recognition sequences. But restriction sites in genomic DNA are polymorphic and exist as two alleles. The RFLP and its position in the genome map can be worked out following the inheritance of its alleles. Polymorphic restriction site. Two methods of RFLP: Southern hybridization. PCR.

9. 6. Simple Sequence Length Polymorphism (SSLP) SSLPs are arrays of repeat sequences that display length variation. Here different alleles contain different numbers of repeat sequences. SSLPs can be multiallelic. Two types of SSLPs are: Minisatellites (VNTRs). 1 Microsatellites are more popular than minisatellites as DNA markers. Microsatellites (STRs). 2

10. 7. Single Nucleotide Polymorphism (SNP) There are some positions in the genome where some individuals have one nucleotide while others have another . Some SNPs give rise to RFLPs but many do not. SNPs originate when a point mutation occurs in the genome converting one nucleotide to another. There are just two alleles - the original sequence and the mutated version. SNPs enable very detailed genome maps to be constructed. These are mainly based on oligonucleotide hybridization analysis: DNA chip technology. Oligonucleotide ligation assay . Solution hybridization . Amplification refractory mutation assay (ARMS test).

11. 8. Demerits 1 Not much sufficient for directing the sequencing phase of a genome project. 2 Limited accuracy. 3 Depends on the number of crossovers that have been scored.

12. 9. Physical Mapping The most important techniques used in physical mapping are as follows: Restriction mapping . Fluorescent in situ Hybridization (FISH). Sequence tagged site (STS) mapping.

13. Restriction Mapping Direct examination of DNA molecules for restriction sites can be done in two ways: Gel stretching. 1 Molecular combing. 2 Restriction mapping is used to find the positions of restriction enzyme sites on a DNA molecule. It is done by cutting the DNA with different restriction enzymes and comparing the sizes of the resulting fragments. By analyzing these fragment patterns, a restriction map showing the relative locations of enzyme recognition sites is created.

14. FISH is a technique used to see the exact position of a gene or DNA marker on a chromosome. It uses a fluorescent probe that binds (hybridizes) to a specific DNA sequence. Under a microscope, the fluorescent signal shows the location of that DNA sequence. In optical mapping, restriction sites appear as gaps on stretched DNA fibers. Fluorescent In Situ Hybridization (FISH)

15. Sequence Tagged Sites (STS) STS mapping is one of the most powerful physical mapping techniques. It helps create detailed maps of chromosomes or genomes. A Sequence Tagged Site (STS) is a short, unique DNA sequence — usually 100 to 500 base pairs long. Each STS occurs only once in the genome, making it easy to identify and locate specific regions of DNA

16. Genetic map Physical map It is constructed using recombination frequency calculated from the progenies. It pertains to locating the position of DNA sequences. It is an indirect method of locating the positions of genes or DNA markers. It is a direct method of locating the positions of genes or DNA markers. Unit of measurement of map distance is cM. Unit of measurement of map distance is base pair. 10. Genetic Map vs Physical Map

17. 11. Importance of Gene Mapping Helps study differences and inheritance patterns in human genetic diseases. Aids in developing methods for gene therapy. Provides useful information for linking genes to diseases. Useful for identifying genetic markers for diagnosis. Assists in understanding how genes affect traits and diseases.

18. 12. Cloning of a Disease Gene Gene cloning: Making multiple copies of a single gene the insertion of a fragment of DNA carrying a gene into a cloning vector and subsequent propagation of recombinant DNA molecules into many copies is known as gene cloning. Disease Gene Cloning: Cloning of a disease gene is the process of isolating and making copies of a gene responsible for a specific disease. Purpose: Helps in studying the gene’s function understanding the disease and developing diagnostic tests or therapies.

19. Cloning of a Disease Gene Methods of Cloning a Disease Gene 1 2 3 Positional Cloning (Map-Based Cloning): Gene is located based on its position on the chromosome using genetic markers. Useful when the gene’s function is unknown. Functional Cloning : Candidate Gene Approach Gene is identified based on its known function or effect in cells. Involves screening DNA libraries to find the gene responsible for the disease. Starts with genes suspected to be involved in the disease. Tests these genes for mutations or abnormal activity.

20. Genetic Testing: Detect carriers and individuals affected by genetic disorders. Gene Therapy: Provides the basis for correcting defective genes in patients. Prenatal Diagnosis: Detect genetic diseases before birth. Research: Study how genes cause diseases and understand their function. Drug Development: Identify new drug targets for treatment of genetic diseases. 13. Applications of Disease Gene Cloning

21. Complex Diseases: Some diseases involve multiple genes, making cloning difficult. Large Genome Size: Locating a single gene in a huge genome can be challenging. Ethical Issues: Manipulating human genes raises moral and ethical concerns. Mutation Variability: Different mutations in the same gene can cause different effects. Technical Limitations: Requires advanced lab equipment and expertise. Challenges of Disease Gene Cloning:

22. 14. Basic Steps of Gene Cloning Construction of recombinant DNA molecule. Multiplication of recombinant DNA molecule. 1 3 Numerous cell divisions resulting in a clone. The gene cloning requires specialized tools and techniques. 5 Transport of the recombinant DNA to the host cell. Division of the host cell. 2 4

23. 15. PCR PCR (Polymerase Chain Reaction) is a laboratory technique used to make many copies of a specific DNA segment. Unlike natural DNA replication in cells, PCR allows scientists to selectively amplify only the gene of interest. In the context of disease gene cloning, PCR is especially useful for copying a disease-causing gene so that it can be studied in detail. The amplified gene can then be analyzed for mutations, used in genetic testing, or serve as a starting point for developing gene therapies. PCR makes it possible to work with very small amounts of DNA, which is often all that is available from patients or samples.

24. Use of PCR in Disease Gene Cloning: Gene Amplification: PCR makes many copies of a disease-causing gene, even from very small DNA samples. Mutation Detection: Amplified DNA can be analyzed to identify mutations responsible for genetic disorders. Diagnostic Testing: PCR allows rapid detection of disease genes in patients for early diagnosis. Research Applications: Facilitates the study of gene function and its role in disease. Gene Therapy Development: Provides sufficient copies of the target gene for experimental gene therapy or cloning experiments.

25. Three Main Stages of PCR Denaturing When the double-stranded template DNA is heated to separate it into two single strands. Annealing When the temperature is lowered to enable the DNA primers to attach to the template DNA. Extending When the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.

26. 16. References Molecular pharmacology: from DNA to Drug discovery. John Dickinson et al.

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