Map based cloning of genome

Introduction
History
Mapping of genome
1. genetic mapping
2. physical mapping
Map based cloning
Steps of MBC
Uses
Conclusion
References

1. Map based Cloning of Genome By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )

2. SYNOPSIS- Introduction History Mapping of genome 1. genetic mapping 2. physical mapping Map based cloning Steps of MBC Uses Conclusion References

3. INTRODUCTION Map based cloning of genome is a method to identify and to make unknown nucleotide sequence.

4. HISTORY- In 1990, clone region around markers, make physical map(s), look for genes experimentally In 2000, use better physical maps, at least in some organisms In 2010, use sequencing, bioinformatic knowledge, experimental proof still necessary

5. MAPPING OF GENOME-

6. TYPES Genetic mapping is based on the use of genetic techniques to construct maps showing the positions of genes and other sequence features on a genome. Physical mapping uses molecular biology techniques to examine DNA molecules directly in order to construct maps showing the positions of sequence features, including genes.

7. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genetic mapping I Based on recombination frequencies The further away two points are on a chromosome, the more recombination there is between them Because recombination frequencies vary along a chromosome, we can obtain a relative position for the loci Distance between the markers

8. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genetic mapping II Genetic mapping requires that a cross be performed between two related organisms The organism should have phenotypic differences (contrasting characters like red and white or tall and short etc) resulting from allele differences at two or more loci The frequency of recombination is determined by counting the F 2 progeny with each phenotype

9. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genetic mapping example I Genes on two different chromosomes Independent assortment during meiosis (Mendel) No linkage Dihybrid ratio F 1 9 : 3 : 3 : 1 F 2 P

10. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genetic mapping example II Genes very close together on same chromosome Will usually end up together after meiosis Tightly linked F 1 1 : 2 : 1 F 2 P

11. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genetic mapping example III Genes on same chromosome, but not very close together Recombination will occur Frequency of recombination proportional to distance between genes Measured in centiMorgans = cM Recombinants Non-parental features One map unit = one centimorgan ( cM ) = 1% recombination between loci

12. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genetic markers Genetic mapping between positions on chromosomes Positions can be genes Responsible for phenotype Examples: eye color or disease trait: limited Positions can be physical markers DNA sequence variation

13. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Physical markers Physical markers are DNA sequences that vary between two related genomes Referred to as a DNA polymorphism Usually not in a gene Examples RFLP SSLP SNP

14. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 RFLP Restriction-fragment length polymorphism Cut genomic DNA from two individuals with restriction enzyme Run Southern blot Probe with different pieces of DNA Sequence difference creates different band pattern GGATCC CCTAGG GTATCC GATAGG GGATCC CCTAGG 200 400 GGATCC CCTAGG GCATCC GGTAGG GGATCC CCTAGG 200 400 * * 200 400 600 1 2 * * 2 1

15. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 SSLP/Microsatellites Simple-sequence length polymorphism Most genomes contain repeats of three or four nucleotides Length of repeat varies due to slippage in replication Use PCR with primers external to the repeat region On gel, see difference in length of amplified fragment ATCCTAC GACGACGACGATT GATGCT 12 18 1 2 2 1 ATCCTAC GACGACGACGACGACGATT GATGCT

16. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 SNP Single-nucleotide polymorphism One-nucleotide difference in sequence of two organisms Found by sequencing Example: Between any two humans, on average one SNP every 1,000 base pairs ATCGATTGCCATGAC ATCGATGGCCATGAC 2 1 SNP

17. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Physical mapping Determination of physical distance between two points on chromosome Distance in base pairs Example: between physical marker and a gene Need overlapping fragments of DNA Requires vectors that accommodate large inserts Examples: cosmids , YACs, and BACs

18. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Restriction mapping applied to large-insert clones Generates a large number of fragments Requires high-resolution separation of fragments Can be done with gel electrophoresis

19. FISH Technique- Fluorescent in situ hybridization In FISH, the marker is a DNA sequence that is visualized by hybridization with a fluorescent probe.   Sequence tagged site (STS) mapping- A sequence tagged site or STS is simply a short DNA sequence, generally between 100 and 500 bp in length

20. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Contigs Contigs are groups of overlapping pieces of chromosomal DNA Make contig uous clones For sequencing one wants to create “minimum tiling path” Contig of smallest number of inserts that covers a region of the chromosome genomic DNA contig minimum tiling path

21. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Contigs from overlapping restriction fragments Cut inserts with restriction enzyme Look for similar pattern of restriction fragments Known as “fingerprinting” Line up overlapping fragments Continue until a contig is built

22. MAP BASED CLONING- What to Do Next? Identify genes in this region Then determine what is the gene of interest

23. Steps of MBC- Identify a marker tightly linked to your gene in a "large" mapping population Find a YAC or BAC clone to which the marker probe hybridizes Create new markers from the large-insert clone and determine if they co-segregate with your gene If necessary, re-screen the large-insert genomic library for other clones and search for co-segregating markers Identify a candidate gene from large-inset clone whose markers co-segregate with the gene Perform genetic complementation (transformation) to rescue the wild-type phenotype Sequence the gene and determine if the function is known

24. Identification a marker-

25. Find a YAC or BAC clone to which the marker probe hybridizes

26. Create new markers from the large-insert clone and determine if they co-segregate with your gene . Promoter Coding Region

27. If necessary, re-screen the large-insert genomic library for other clones and search for co-segregating markers.

28. Identify a candidate gene from large-inset clone whose markers co-segregate with the gene .

29. Perform genetic complementation (transformation) to rescue the wild-type phenotype .

30. Map-based Cloning 1. Use genetic techniques to find marker near gene Gene Marker 2. Find cosegregating marker Gene/Marker 3. Discover overlapping clones (or contig) that contains the marker Gene/Marker 4. Find ORFs on contig Gene/Marker 5. Prove one ORF is the gene by transformation or mutant analysis Mutant + ORF = Wild type? Yes? ORF = Gene

31. USES- To make resistance in plants and animals. Diagnosis in diseases. To make vaccines.

32. CONCLUSION MBC is a new technology to create a clone of necessary nucleotide sequence. It can be modified and changed , which depends on the type of the species.

33. REFERENCES ta brown ,Genomes ,2nd edition Watson , Molecular Biology of the Gene (5th edition, 2004) http://www.inia.org.uy/