Genome mapping

What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd

1. GENOME MAPPING : fish RFLP & RApd PRESENTED BY- RASHMI YADAV

2. What is a Genome ? The haploid set of chromosomes in a gamete or microorganisms, or in each cell of multicellular organisms. It is encoded either in DNA or for many types of viruses, in RNA. Each genome contains all of the information needed to build and maintain that organisms.

3. Genome mapping “ A graphic representation of the arrangement of genes or DNA sequences on chromosome”. The human genome map completed in 1996 locates 5264 markers for gene. A genome map are used to identify and record the location of gene & distances between genes on chromosome . Genome mapping by Fluorescent fingerprinting

4. Types of genome mapping The two main kinds of genome maps are known ; Genetic or linkage maps & Physical maps Both maps are a collection of genetic markers

5. linkage or genetic mapping Genetic maps illustrate the order of genes on a chromosomes and the relative distances between those genes. Genetic maps are based on recombination, the exchange of DNA sequences between non-sister chromatids during meiosis.

6. A genetic map is prepared on the basis of recombination data between carefully selected genetic markers usually ordered into suitable crosses. Recombination frequency= No. of recombinant progeny 100 % Total No. of progeny

7. But in case of humans, linkage maps have to be prepared using family pedigree data. In such maps, the distances between genes are shown in terms of map units or centiMorgans ( cM ). The chief problem of linkage mapping is the non availability of a sufficient number of genetic markers to cover the entire genome.

8. Physical mapping A Physical map provides detail of the actual physical distance between genetic markers, as well as the exact location of genes . There are three most important techniques used to create a physical map : Somatic cell hybridization Radiation hybridization Fluorescent in situ hybridization

9. Somatic cell hybridization Also called somatic cell fusion or protoplast fusion . Development of hybrid plants through the fusion of somatic protoplasts of two different plant species is called somatic hybridization. Somatic hybridization involves the following 4 steps; Isolation of protoplast. Fusion of the protoplasts of the desired species. Selection of somatic hybrid cells. Culture of the hybrid cells and regeneration of the hybrid plants from them.

10. Somatic cell hybridization Medium HAT

11. is a method for high resolution mapping. In radiation hybrid mapping uses radiation such as x- ray , to break the DNA into fragments. The amount of radiation can be adjusted to create smaller or larger fragments. This technique is not affected by increased or decreased recombination frequency. Radiation hybridization

12. Fish ( =fluorescence in - situ hybridization) FISH is a powerful technique for detecting RNA or DNA sequences in cells, tissues & tumors. FISH provides a unique link among the studies of cell biology , cytogenetics , & molecular genetics . FISH allows very precise spatial resolution of morphological & genomic structures. The technique is rapid, simple to implement and offers great probe stability .

13. Types of probes for FISH Locus specific probes – binds to a particular region of a chromosome. This type of probe is useful when researchers have isolated a small portion of gene and want to determine on which chromosome the gene is located. Alphoid or centromeric repeat probes – are generated from repetitive sequences found in the middle of each chromosome Whole chromosome probes - are actually collections of smaller probes, each of which binds to a different sequence along the length of a given chromosome.

14. applications FISH has a large number of applications in molecular biology and medical science including; Gene mapping Diagnosis of chromosomal abnormalities Studies of cellular structure and function In clinical research ; Prenatal diagnosis, Cancer diagnosis Molecular cytogenetic of birth defects & mental retardation Diagnosis of infectious diseases & Detection of aberrant gene expression

15. In laboratory research FISH can be used for; To study the evolution of genomes Analyzing nuclear organization Visualization of chromosomal territories and chromatin in interphase cells etc. Chromosome painting –

16. Molecular markers In genetics, a molecular marker is a fragment of DNA that is associated with a certain location within the genome. Molecular markers are used to identify a particular sequence of DNA in a pool of unknown DNA. Isozymes ( electrophoretic variants of enzymes) and DNA sequences are used as molecular markers in chromosome mapping.

17. A molecular genetic marker may can be divided into two classes ; An Biochemical marker which detect variation at the gene product level such as changes in proteins and amino acids . Molecular markers which detect variation at the DNA level such as Nucleotide changes , Deletion , Duplication .

18. Rflp (= Restriction fragment length polymorphism) A RFLP is a genetic marker that can be examined by cleaving the DNA into fragments with a restriction enzymes. A restriction enzyme cuts the DNA molecule at every occurrence of a particular sequences called restriction site . For example, HindII enzyme cuts at GTGCAC or GTTAAC. If we apply a restriction enzyme on DNA, it is cut at every occurrence of the restriction site into a million restriction fragments each a few thousands nucleotide long.

19. RFLPs may have the following Applications; Identification and isolation of any gene known to be linked with an RFLP locus. In paternity cases or criminal cases to determine the sources of DNA sample. Identification of the most important loci affecting a quantitative trait. Determination of chromosome segments alteration of which is likely to yield the best results. Diagnostic in genetically inherited disease. Identifying hybrids . Pedigree analysis . Genetic screening.

20. Advantages of rflp

21. disAdvantages of rflp Expensive Requires relatively large amount of DNA. Low levels of polymorphism in some species. Need a suitable probe library. Time consuming especially with single copy probes. Costly

22. Rapd (= Random amplification of polymorphic DNA) It is a PCR based technology. In 1991 Welsh and Maclelland developed this technique. This procedure detects nucleotide sequence polymorphism in DNA. It detects dominant variation in the genome. It is used to analyse genetic diversity of an individual by random primers .

23. Process of rapd Primer Agarose gel Mutation DNA amplified products DNA segments Small DNA segment H-1 genome H-2 genome

24. Gene mapping DNA amplification finger printing. Study of closely related species. applications ADVANTAGE They are quick and easy to assay, because PCR is involved. Only low quantities of template DNA are required. RAPD have a very high genomic abundance and are randomly distributed throughout the genome. Pure DNA is not needed.

25. disADVANTAGE of rapd Markers are dominant i.e. they can’t distinguish whether a DNA sequence is amplified from a locus that is homozygous or heterozygous. PCR is an enzymatic reaction, therefore, the quality and concentration of template DNA, conc. of PCR components and the PCR cycling conditions may greatly influence the outcome. Mismatches between the primer and the template may results in the total absence of PCR product as well as merely decreased amount of the product. Thus, the RAPD results can be difficult to interpret.

26. Difference between rflp & rapd RFLP RAPD 1. Large quantity of purified DNA required. 1. Quantity of DNA required for analysis is small. 2. Different species specific probes are required. 2. Same primers with arbitrary sequence can be used for different species 3.Comparatively slower processing due to more steps involved. 3. Fewer steps in procedure therefore it is rapid (Five times quicker than RFLP) 4. Technique comparatively more reliable 4. Technique comparatively less reliable

27. 5. Can detect allelic variants 5. Cannot detect allelic variants 6 . 1-3 loci detected 6. 1-10 loci detected 7. Method involves:        a) digestion of extracted DNA by restriction enzymes,        b) gel electrophoresis of fragments,        c) southern blot by specific probes and detection of specific sequences 7. Method involves:        a) extraction of DNA,        b) amplification by PCR using random primers,        c) gel electrophoresis of amplified DNA and visualization of markers RFLP RAPD

28. references Genome mapping – Wikipedia the free encyclopedia Biotechnology – B. D. Singh Genetics principle and analysis – Daniel L. Hartl - Elizabeth W. Jones Biotechnology – S. S. Purohit Biotechnology (Applying the genetic revolution)- David P. Clark & Nanette J. Pazdernik

29. Thank you