Transgenic Plants, Gene Identification and Method.pptx

The importance of transgenic plants, gene identification and methods of gene identification.

1. T ransgenic Plants, Gene Identification Methods & Localization and Sequencing of Genes By: Annu  M. Pharmacy [DRA 2nd Sem] 2239807

2. Transgenic Plants

3. Definition 

4. History 

5. Production of Transgenic Plants  It can be carried out by several methods such as: Particle Gun or Gene Gun or Biolistic or microprojectile bombardment. PEG (polyethylene glycol mediated transformation) Electroporation  Agrobacterium –mediated gene transfer.

6. Particle gun/ Particle bombardment   Foreign DNA coated with high velocity gold or tungsten particles to deliver DNA into cells. This method is widely being used because of its ability to transfer foreign DNA into the mammalian cells and microorganisms.

7. Polyethylene glycol-mediated transformation PEG in the presence of divalent cations, destabilizes the plasma membrane of protoplasts and renders it permeable to naked DNA.  A large number of protoplasts can be simultaneously transformed. This technique can be successfully used for a wide variety of plant species.

8. Electroporation  It involve the creation of pores in the cells membrane using electric pulse of high field strength. If DNA is present in the buffer solution at sufficient concentration, it will be taken up through these pores.

9. Agrobacterium mediated gene transfer It is treated as nature's most effective plant genetic engineer. A tumefaciens infects wounded or damaged plant tissues results in the formation of plant  tumor called crown gall. The bacterium releases  Ti plasmid into the plant cell cytoplasm which induce crown gall. Several dicots are affected by this diseases e.g. grapes, roses etc.

10. Importance of transgenic plants

11. Methods used in gene identification

12. Gene identification   Identification of important components in genomic DNA. Identification of genes in a genomic DNA Sequence. Prediction of protein-coding genes: Prokaryotes  Unicellular eukaryotes Multicellular eukaryotes

13. Gene identification methods Genes are identified broadly via two methods: Similarity-based searches Ab-initio prediction

14. Similarity-based Searches This method of gene identification is based on sequence similarity searches. Similar genetic sequences are found between ESTs (expressed sequence tags), proteins, or other genomes. This method assumes that exons (functional regions) are conserved evolutionarily than introns (non-functional region). Commonly used bioinformatics tool that is based on the similarity search method is BLAST. Other commonly used software are PROCRUSTES and GeneWise .

15. Ab-initio Prediction This method of gene identification is based on gene structure and signal-based searches. Ab-initio gene predictions used known gene structure as a template to determine unknown genes.  This method is based on two types of sequence information, namely, signal sensor and content sensors. Models of gene prediction are: FGENESH, GENEID, GENEPARSER, GENESCAN, GLIMMER M etc.

16. Localization & Sequencing of genes

17. Gene sequencing

18. Gene sequencing methods: First generation sequencing: Sanger sequencing Maxam Gilbert sequencing 2.    Second generation sequencing 3.    Next generation sequencing: Template preparation Library preparation Library amplification

19. First generation sequencing: Sanger Sequencing: This method is used in in-vitro DNA replication. This technique is developed by British Biochemist Frederick and his team in 1977.

20. Maxam Gilbert Sequencing: This technique developed by Allan Maxam and Walter Gilbert in 1976. Principle: Chemical degradation of Pyrimidines.

21. Second Generation Sequencing:

22. Next generation sequencing: Basic Principle: Its works is similar to traditional Sanger Sequencing methods involving capillary electrophoresis. Template preparation : The double-stranded DNA is considered as the starting material. However, the source from which this material is derived may vary. Sources can be either genomic DNA, immuno-precipitated DNA, reverse-transcribed RNA or cDNA. Library preparation : Sequence library preparation involves some common steps of fragmentation, size selection and adapter ligation. Adapter ligation is also involved in this process, which adds platform specific, synthetic DNAs at the end of the DNA fragments present in this library to facilitate the sequencing reactions.

23. 3.  Library amplification : to produce significant signal for nucleotide addition. This step involves either by the attachment of DNA fragment to micro bead or attachment of the same to glass slide, when some PCR techniques are followed. 

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