Paper No. : 16 Molecular Genetics Module : 21 Methods for gene identification: In-situ Hybridization Development Team Principal Investigator: Prof. Neeta Sehgal Head, Department of Zoology, University of Delhi Co-Principal Investigator: Prof. D.K. Singh Department of Zoology, University of Delhi Paper Coordinator: Prof. Namita Agrawal Department of Zoology, University of Delhi Content Writer: Dr. Jasvinder Kaur, Dr. Poonam Sharma, Gargi College, University of Delhi Content Reviewer: Dr. Surajit Sarkar Department of Genetics, South Campus, University of Delhi 1 ZOOLOGY Molecular Genetics In-situ Hybridization Description of Module Subject Name ZOOLOGY Paper Name Zool 016: Molecular Genetics Module Name/Title Methods for gene identification Module ID M21: In-situ Hybridization Keywords In-situ Hybridization, Probe, Radioactive label, Fish, Chromosome painting Glossary Avidin: Biotin binding protein, found in the egg whites of birds, amphibians and reptiles (produced in their oviducts). Chromosome mapping: Technique using which genes belonging to same gene families can be detected by hybridization with suitable labeled probes. Chromosome painting: Multiple fluorochromes can be used in FISH technique which produces a multicolored and painted effect with unique color at each hybridization site in the chromosomes being studied giving the effect of chromosome painting. Fluorescent in-situ hybridization:It is a cytogenetic technique that uses fluorescent probes to detect the presence or absence of specific DNA sequences on chromosomes. Hybridization probe:Probe is a fragment of DNA or RNA of variable length, which can be radioactively labelled. Used to detect nucleotide sequence, complementary to the sequence in the probe. Immunohistochemistry: Technique used to localize proteins such as antibodies in tissue sections by antigen-antibody interactions. In-situ hybridization (ISH): ISH is a technique used to detect and localize specific mRNA sequences (within preserved tissues) and cells (within a heterogeneous cell population).It is based on the complementary base pairing of labelled cDNA, or, RNA probe to normal or abnormal nucleic acid sequences in chromosomes, cells or tissues. Multiple ISH: This technique uses more than one probe to detect different nucleic acid targets in the same tissue section. Oligonucleotide: Short DNA or RNA molecules having wide range of applications. Polymerase chain reaction ISH: This is a variable ISH technique used for detection of targets present even in low copy numbers. 2 ZOOLOGY Molecular Genetics In-situ Hybridization Probes: Complementary sequence of nucleotide bases to the specific sequence of interest such as mRNA are referred to as probes. Riboprobes: RNA probes, 50-1000 bp in length. RNA-RNA hybrid probes are very thermostable and are resistant to digestion by RNases. Q-FISH: This technique uses FISH and computer software to quantify fluorescence intensity. References 1. Meng, L., and Leng, P. In situ hybridisation: principles and applications. 1992. Malaysian J Pathol. 14(2): 69 – 76. 2. Clare O'Connor. Fluorescence in situ hybridization (FISH). 2008. Nature Education 1(1):171 Weblinks: http://www.powershow.com/view/1b405eNzBkN/In_Situ_Hybridization_powerpoint_pp t_presentation 3. http://www.genedetect.com/insitu.htm 4. https://en.wikipedia.org/wiki/In_situ_hybridization 5. http://www.ncbi.nlm.nih.gov/probe/docs/techish/ Further Readings: 1. Fluorescence In Situ Hybridization (FISH) - Application Guide. Edited by Thomas Liehr 2. In Situ Hybridization: Principles and Practice.Julia M. Polak, James O'Donnell McGee.Oxford University Press, 1998 3. In Situ Hybridization: A Practical Guide.A. R. Leitch.BIOS Scientific Publishers, 1994. 3 ZOOLOGY Molecular Genetics In-situ Hybridization .