Application of molecular probes
- 1. APPLICATION OF MOLECULAR PROBES PRESENTED BY AYUSH JAIN PALB 7286
- 2. CONTENTS Molecular Probes In Evolutionary Studies Molecular Probes In Linkage and Chromosomal Mapping Molecular Probes in Molecular Cytogenetics Molecular Probes in DNA Fingerprinting. Conclusion Bibliography January 11, 2018PRINCIPLES OF BIOTECHNOLOGY (PBT 501) 2
- 3. Molecular Probes In Evolutionary Studies January 11, 2018PRINCIPLES OF BIOTECHNOLOGY (PBT 501) 3
- 4. • Genomes has evolved by processes like duplication and accumulation of useful mutations into a common ancestral sequences. • This knowledge can be used to find out evolutionary relationship between the two organisms. • As alterations has taken place both in expressed and unexpressed regions in genome RFLP gives fragments of different length. • RFLP coupled with DNA probe (a common sequence from Ancestor) can be used to find out the divergence and relativeness among the compared organisms. Courtesy: C. T. Federici á D. Q. Fang R. W. Scora á M. L. Roose Phylogenetic relationships within the genus Citrus (Rutaceae) and related genera as revealed by RFLP and RAPD analysis January 11, 2018PRINCIPLES OF BIOTECHNOLOGY (PBT 501) 4
- 5. Molecular Probes In Linkage and Chromosomal Mapping January 11, 2018PRINCIPLES OF BIOTECHNOLOGY (PBT 501) 5
- 6. • Linkage can define the genetic distances between polymorphic traits which may be recognized as differences in appearance of enzyme activities, restriction fragment lengths or nucleotide sequences at an allelic locus. • Low number of morphological markers are available to the plant breeder for crop improvement programs and the expression these morphological markers is affected by environmental conditions which is not the case with molecular markers. • It has application in identification of genetic disorders like sickle cell anaemia, cystic fibrosis etc. and also in MAS Breeding. January 11, 2018PRINCIPLES OF BIOTECHNOLOGY (PBT 501) 6
- 7. Marker Assisted Selection Breeding • One of the major Application of molecular mapping is MAS Breeding. • Molecular markers are specially advantageous for agronomic traits that are otherwise difficult to tag such as resistance to pathogens, insects and nematodes, tolerance to abiotic stresses, quality parameters and quantitative traits. PRINCIPLES OF BIOTECHNOLOGY (PBT 501) January 11, 2018 7 Characters Examples Rice Resistance to blast caused by Pyricularia oryzae Pi-2(t) gene located 2.8 cM from RG64 on chromosome 6 Pi-4(t) gene located 15.3 cM from RG 869 on chromosome 12 Pi-10t gene tagged with RAPD markers RRF6 and RRH18 on chromosome 5. Resistance gene Xa21 to bacterial blight caused by Xanthomonas oryzae Located 5.3 cM away from RAPD marker RAPD818, RAPD248 and RG103 Co-segregated xa5 with RFLP markers RZ390, RG556, RG207 on chromosome 5. Brassica Resistance against Leptosphaeria maculans Resistance against Albugo RFLP markers on linkage group 6 in Brassica napus RFLP markers on linkage candida in B. napus group 9
- 8. Molecular Probes in Cytogenetics • Isolation of genes • In-situ Hybridization January 11, 2018PRINCIPLES OF BIOTECHNOLOGY (PBT 501) 8
- 9. Isolation of Genes • Specific Molecular Probes Can be Used For Isolation of Specific Genes. • Probes if Used from same Species are Called Homologous Probes and From other Species are called Heterologous Probes. • Heterologous Probes are found Effective in Identifying Genes Cloned during colony Hybridization. • These Probes are used with c-DNA Library and not with Genomic Library as it can Harbour Unrelated or Pseudogenes. • Example: CHS (Chalcone Synthase gene from Antirrhinum majus and Petunia hybrida were isolated using c-DNA probe and was used to isolate CHS gene from Barley. PRINCIPLES OF BIOTECHNOLOGY (PBT 501) January 11, 2018 9
- 10. In-Situ Hybridization It is a Technique which permits detection of DNA and RNA sequences in Cell Smears, Tissue Sections and Metaphase Chromosome spreads. It is based on the Formation of Double Stranded hybrid molecules between Target sequence and Complimentary single stranded Labelled probe. Telomeres have been Identified in Human and other Eukaryotes using Telomeric sequence as a Molecular probe showing that telomeres of all carry the same sequence. This method is particularly useful if Target sequences are distributed in a non-random way in tissues for visualization of heterogeneity and study of cell differentiation. Used for rapid screening of small numbers of cultured cells for expression of Oncogenic m-RNA. PRINCIPLES OF BIOTECHNOLOGY (PBT 501) January 11, 2018 10
- 11. Molecular Probes in DNA Fingerprinting. PRINCIPLES OF BIOTECHNOLOGY (PBT 501) January 11, 2018 11 Invented by Sir Alec Jeffreys in 1985.
- 12. • It is also known as Genetic fingerprinting or DNA profiling. • DNA profiling, based on typing individual highly variable minisatellites called VNTR’s (Variable Number of Tandem Repeats) in the human genome. • VNTR’s can contain anywhere from 20- 100 bp. • A southern blot is performed and then probed through a hybridization reaction with a Radioactive version of VNTR in question. • DNA fingerprinting has a high success rate and a very low false positive rate making it an extremely popular form of paternity and maternity verification. PRINCIPLES OF BIOTECHNOLOGY (PBT 501) January 11, 2018 12
- 13. Uses of DNA Fingerprinting Pedigree analysis and establishing paternity. Immigration authorities. Identification of mutilated dead bodies. Social security record identification. In characterization of cell cultures. In animal breeding programmes. In plant breeding programmes. PRINCIPLES OF BIOTECHNOLOGY (PBT 501) January 11, 2018 13
- 14. PRINCIPLES OF BIOTECHNOLOGY (PBT 501) January 11, 2018 14 In demographic studies. Twin zygosity determination Tissue culture cell line identification Mopping fine grained sequence variation in mini satellites.
- 15. CONCLUSION • Since Molecular probes assays are most sensitive than conventional diagnostic methods, their use has become today‘s most sophisticated and sensitive technology for a variety of uses involving biological systems both in basic and applied studies. • They give new dimensions to concerted efforts of breeding and marker-aided selection that can reduce the time span of developing new and better varieties • This ideal technology would offer absolute specificity, modularity, minimal size, deliverability (access to all cell types) and physiological neutrality (noncytotoxic, biochemically) PRINCIPLES OF BIOTECHNOLOGY (PBT 501) January 11, 2018 15
- 16. BIBLIOGRAPHY • Anderson J A, Sorrells M E and Tanksley S D (1993), “RFLP Analysis of Genomic Regions Associated With Resistance to Preharvest Sprouting in Wheat”, Crop Sci., Vol. 33, pp. 453-459. • Deshpande A D, Ramakrishna W, Mulay G P, Gupta V S and Ranjekar P K (1998), “Evolutionary and Polymorphic Organization of the Knotted-1 homeobox in Cereals”, Theor Appl Genet., Vol. 97, pp. 135-140 • Fukuchi A, Kikuchi F and Hirochika H (1993), “DNA fIngerprinting of Cultivated Rice With Rice Retrotransposon Probes”, Jpn. J Genet., Vol. 68, pp. 195-204. • Madan Mohan, Suresh Nair, A. Bhagwat, T. G. Krishna, Masahiro Yano ,C.R. Bhatia, Takuji Sasaki “Genome mapping, molecular markers and marker-assisted selection in crop plants”, Mol. Breed., April 1997, Volume 3, Issue 2, pp 87–103 • K Vasavirama “MOLECULAR PROBES AND THEIR APPLICATIONS”, Review Article, Int. J. LifeSc. Bt & Pharm. Res. 2013, Vol. 2, No. 2, April 2013 PRINCIPLES OF BIOTECHNOLOGY (PBT 501) January 11, 2018 16
- 17. A Tribute to Molecular Biologist all time on his 96th Birthday • He Deciphered the Genetic Code • 1968 Nobel Prize for Physiology or Medicine. • Dr. Har Gobind Khorana PRINCIPLES OF BIOTECHNOLOGY (PBT 501) January 11, 2018 17