Microbial biotechnology SlideShare, DNA LIBRARIES

Microbial biotechnology DNA LIBRARIES

1. Submitted by , Nathiya Kumar, III Bsc microbiology , Department of microbiology , VIAAS, Sankagiri . Subject incharge , R.PANDIAN, Assistant professor, Department of microbiology VIAAS, Sankagiri VIVEKANANDHA Arts and science college for women, veerachipalayam sankagiri – 637303, Salem Dt., Tamilnadu , India. Affiliated with periyar university , Salem Recognised Under section 2(f) & 12(b) , UGC ACT 1956. SUBJECT: MICROBIALBIOTECHNOLOGY TOPIC: DNA LIBRARIES

2. Welcome DNA LIBRARIES DNA LIBRARIES

3. DNA LIBRARY Gene libraries or DNA libraries are collections of cloned genes that are big enough to contain at least one copy of every gene from a particular organism. The size of the genes and the organism dictate which vector is used for holding the inserts. The genes of prokaryotes are relatively short, averaging about 1000 bp each. In contrast, eukaryotic genes are much longer, largely due to the presence of introns. Different strategies must therefore be followed for prokaryotic and eukaryotic gene libraries as discussed below. Gene libraries may also be made from environmental DNA samples. Such metagenomic libraries include genes from multiple organisms found in a particular environment.

Collections of cloned genes carried in vectors are called libraries. DNA libraries have all the genes from one organism, whereas metagenomic libraries have genes from multiple organisms that inhabit a particular environment.

4. To make a prokaryotic gene library, the complete bacterial chromosomal DNA is cut with a restriction enzyme and each of the fragments is inserted into a vector, usually a simple ColE1-derived plasmid (Fig. 7.17). This mixture of vectors containing a different piece of the bacterial chromosome is transformed into a suitable bacterial host strain and a large number of colonies, each containing a single vector plus insert, are kept. These must then be screened for the gene of interest. If the gene has an observable phenotype, this may be used. Otherwise, more general methods such as hybridization or immunological screening are necessary.

5. cDNA Library cDNA libraries have been broadly used to determine the expressed portion of protein-coding genes in eukaryotes. The construction of a cDNA library involves the extraction and purification of mRNA (Fig. 2.8). These mRNAs are used as a template for the synthesis of cDNA by the process of reverse transcription in the presence of oligo dT primer. The oligo dT primer binds with the poly-A tail of mRNA followed by synthesis of the first strand of cDNA by using reverse transcriptase enzyme.

6. After the synthesis of the first strand, mRNA is removed from DNA: RNA hybrid with the help of RNAse enzyme leaving a single-stranded cDNA. This single-stranded cDNA has the tendency to form a hairpin loop at the 3ʹ end, which provide 3′ hydroxyl group for second-strand synthesis (self-priming). The single-stranded cDNA is converted into a double-stranded DNA with the help of DNA polymerase. After the synthesis of the second strand, the loop at 3′ end is opened by the action of single-strand-specific S1 nuclease. The synthesized cDNA is further cloned into a suitable vector, followed by the transformation of recombinant vectors into a suitable host to create a cDNA library. cDNA libraries contain only the actively transcribed genes of an organism. The cDNA libraries lack information about enhancers, introns, and other regulatory elements because cDNA is synthesized from fully transcribed and processed mRNA. Introns would pose a problem when the eukaryotic gene is expressed in bacteria because most bacteria do not have any mechanism for the removal of introns. cDNA can be promptly expressed in a bacterial cell because mature mRNA is already spliced in eukaryotic cells, and hence the produced cDNA lacks introns.

7. Genomic DNA LIBRARY Construction (Preparation) of Genomic Library
1. Isolation of Genomic DNA
The construction of a genomic library begins with the isolation of genomic DNA from the organism of interest.
Genomic DNA can be isolated using different methods such as cell lysis, protein digestion, and phenol-chloroform extraction.
The isolated DNA represents the entire genome of the organism and contains both coding and non-coding regions.

8. Fragmentation of Genomic DNA
The isolated genomic DNA obtained is often too large to be cloned directly into vectors. So, it needs to be fragmented into smaller fragments suitable for cloning.
This fragmentation can be achieved using physical methods such as sonication, mechanical shearing, or enzymatic methods involving restriction enzymes.

9. Cloning
The fragmented genomic DNA is then cloned into a suitable vector. Some common vectors used for genomic library construction include plasmids, bacteriophages, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs).
Bacterial vectors are suitable for smaller DNA fragments, while YACs are used for larger DNA fragments.

10. The vectors are treated with restriction enzymes to create sticky ends that are compatible with the fragmented DNA.
DNA ligase enzyme is used to bind the DNA fragments to the vector, creating recombinant DNA molecules.

11. Transformation
The recombinant vectors containing the cloned genomic DNA fragments are transformed into a suitable host organism, usually E. Coli and yeast.
The transformed host cells take up the recombinant vectors and are cultured on agar plates containing selective media to allow the growth of colonies containing the recombinant DNA. These colonies form a genomic DNA library.

12. Genomic DNA LIBRARY