chromosome micro dissection nd micro cloning.pptx

Chromosome micro dissection and chromosome micro cloning technique

1. JAI NARAIN VYAS UNIVERSITY, JODHPUR DEPARTMENT OF BOTANY DCC-BOT 8022T – CYTOLOGY AND GENETICS TOPIC – CHROMOSOME MICRODISSECTION AND MICROCLONING SUBMITTED TO :- DR.BHANARAM GADI SUBMITTED BY :- DIKSHA SHARMA (M.Sc. PREVIOUS)

2. Index 1. Introduction 2. Materials required and applications 3. Comparisons of main techniques in the plant chromosome 4. Experimental procedures 5. References

3. INTRODUCTION Chromosome microdissection and microcloning is a technique that acts as a bridge between cytogenetics and molecular genetics. It has been widely applied and play an important role in many studies, such as chromosome painting probe isolation, genetic linkage map, and physical map construction, and expressed sequence tags generation . Chromosome microdissection and microcloning , a technique in which the target chromosome/segment is cut or burned by glass micro needles or lasers in an inverted microscope for separation from other chromosomes and the isolated chromosome/segment is then collected and amplified in vitro to establish specific DNA libraries. This technique was developed and first used to cut the chromosomes of Drosophila melanogaster by Scalenghe et al in1981. However , it is still mainly applied to studies of human and animal chromosomes. The application of chromosome micro dissection in plant research is limited because ( 1) the thick cell wall, denser cell cytoplasm, and asynchronous cell division make chromosome preparation difficult in plants and ( 2) chromosome banding is not perfect in most plants, so it is very difficult to distinguish the target chromosome. This technique of chromosome microdissection and microcloning was successfully used in rye chromosomes by Saudery et al. (1991), and the cloning of rye DNA was successfully achieved.

4. The advantage of chromosome microdissection is that any chromosome or DNA marker can be accurately obtained for any chromosome region according to the research need. Applications:- 1. An efficient and direct approach for isolating DNA from specific chromosomes and/or specific sections. 2. The isolated DNA is used for genomic research including- a). Genetic linkage map and physical map construction. b). Generation of probes for chromosome painting. c). Generation of chromosome specific expressed sequence tags. Materials required:- 90% acetic acid, 70% ethanol, DOP PCR primer, dNTP , Taq polymerase, nitrous oxide. DNA polymerase I, etc.

5. FLUORESCENT IN-SITU HYBRIDIZATION {FISH} It is a laboratory technique for detection and locating a specific DNA sequence in metaphase or interphase cells. Fluorescent dyes are utilized to label the probe. Detection is done with the help of fluorescent microscope. Application- To detect chromosomal abrerations or diagnosis of genetic disorders. Steps involved in FISH- Preparation of sample Denaturation Probe hybridization Washing Detection Analysis

6. ADVANTAGES AND DISADVANTAGES OF FISH ADVANTAGES Can be applied to both dividing and non-dividing cells. Simultaneous detection with multiple probes. For studies of chromosomal changes and gene mapping. DISADVANTAGES Cannot detect small mutations. Probes are not yet commercially available.

7. MULTICOLOURED FISH TECHNIQUE {M-FISH} Labelling of probes to multi- colour detection and the creation of multi- colour FISH images. Multi-probe detection increases the speed of chromosome analysis. Multicolor FISH (M-FISH) assays are used for a precise assessment of complex chromosomal rearrangements. This technique uses all whole-chromosome painting probes in multiplex-FISH and spectral karyotyping . Thus,  marker chromosomes, complex chromosomal rearrangements, and all numerical aberrations  can be visualized simultaneously in a single hybridization experiment.

8. REFERENCES Jiang CZ, Song WQ, Li XL, Chen RY(1998). Studies on microdissection and microcloning of the rye chromosome 1R. Acta Bot Sin 40:988-993. Zhon RN, Hu ZM (2007). The development of chromosome microdissection and microcloning technique and its applications in genomic research. Curr Genomics 8:67-72. Albani , D., Cote, M.J., Armstrong; K.C. Chen, Q., Segal, A. and Robert, L.S. 1993. PCR amplification of microdissected wheat chromosome arms in a simple single tube reaction. Plant J. 4:899-903. Maoyin Sheng , Mengdi Gao , Linjiao Wang and Xuelian Ren . 2020. Chromosome Microdissection and Microcloning : Technique and Application in the Plant Sciences. Ying- Xin Zhang , Chuan-Liang Deng , and Zan -Min Hu . 2016. The Chromosome Microdissection and Microcloning Technique. Plant Cytogenetics : Methods and Protocols pp. 151-160.

9. THANK YOU