Gene library

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Gene library

  1. 1. Presented by D.Priya BSA-11-444 & J. Delince BSA-11-409
  2. 2. Gene Library and Types  A large collection of DNA fragments cloned from a given organism, tissue, organ, or cell type. It may contain entire genomic sequences or complementary DNA sequences formed from messenger RNA .
  3. 3.  For the construction of Gene libraries we have to know the size of the gene and the insert size or capacity of the vector. So that we can determine the size of Gene library and also to ensure the high probability of the insertion.  Also the molecular tools like Restriction enzymes( Molecular scissors) and ligases( Molecular paste) were used .  Vectors like Plasmids, Lambda phage, cosmid, BAC or YAC( yeast artificial chromosome) vectors can be used to construct genomic libraries, the choice depending on the genome size.
  4. 4. Vectors , Insert size, Clone requirement Vector type Maximum insert size Cloned DNA (kb) Approx. No. of clones required in library Plasmid ~20 kb >105 lambda phage 20 kb 5 x 105 Cosmid 45 kb 2 x 105 BAC (bacterial artificial chromosome) > 500 kb 5 x 104 YAC (yeast artificial chromosome) 1 Mb 105
  5. 5. Genome:  It is the totality of DNA sequences present in an organism  This includes the DNA of Chloroplast and Mitochondria  The genome size of an individual is expressed in terms of number of base pairs either in kilobases (1000 bp) or in megabases (one million bp).  The smallest genome about 130mbp for Arabidopsis thaliana (thale cress)  For us the genome size- 3300mbp
  6. 6. Genomic DNA Libraries • These libraries are made from genomic DNA Genomic DNA molecules are very large , so they must be fragmented into small pieces to insert into vectors. • This is done through digestion using one or more appropriate restriction endonucleases. • The DNA is then ligated into the vector, which could be a plasmid, a cosmid or in a Phage
  7. 7. Construction of Genomic Libraries • The construction of a genomic library begins with cleaving the genome into small pieces by a restriction endonuclease. • These genomic fragments are then either cloned into vectors & introduced into a microbe or packed into phage particles and assembled that are used to infect the host. • In either case, many thousands of different clones- each with a different genomic DNA insert –are created. • Therefore each clone will act as a “book” in this “library” of DNA fragments. • If the genomic library has been inserted into a microbe that expresses the foreign gene, it may be possible to assay each clone for a specific protein or phenotype.
  8. 8. Genomic library
  9. 9. Screening of the Genomic Library  The libraries were screened to identify the genes of interest.  For example: finding the gene for alanine production in a bacterium we have to host genomic sequence of that bacterium in E.coli  Then they were grown in alanine deficit medium to remove the clone having other genes  They were labeled in the petri plate which indicates that the gene for alanine production is stored in the bacteria.  This method is called expression screening  Or we can use probes for screening(hybridization technique)  Probes may be a oligonucleotide or short segment of DNA which may be labeled or taken from related species  These probes are hybridized with the DNA in the Nylon membrane and visualized in UV or X-ray.  Or we can do the blotting methods for checking the prescence of gene of interest
  10. 10. cDNA Libraries  These libraries are made from cDNA (complimentary DNA), which are DNA copies from mRNA molecules.  To make cDNA, mRNA is isolated from a tissue or whole organism, and DNA is copied from the mRNA template using an enzyme called reverse transcriptase.  This enzyme uses the reverse genetics principle and found in retrovirus.  The resulting cDNA molecules are then engineered so that they have restriction enzyme recognition sites at each end of every molecule, which allows them to be digested and inserted into a vector
  11. 11. mRNA extraction  The mRNA was extracted and purified by trizol extraction and column purification.  Column purification is done by using oligomeric dT nucleotide coated resins where only the mRNA having the poly-A tail will bind.  The rest of the RNAs are eluted out.  The mRNA is eluted by using eluting buffer and some heat to separate the mRNA strands from oligo-dT.
  12. 12. cDNA construction • Once mRNA is purified, oligo-dT (a short sequence of deoxy-thymine nucleotides) is tagged as a complementary primer which binds to the poly-A tail providing a free 3'-OH end that can be extended by reverse transcriptase to create the complementary DNA strand. • Then the mRNA is removed by using a RNAse enzyme leaving a single stranded cDNA (sscDNA). This sscDNA is converted into a double stranded DNA with the help of DNA polymerase. However, for DNA polymerase to synthesize a complementary strand a free 3'-OH end is needed. This is provided by the sscDNA itself by generating a hairpin loop at the 3' end by coiling on itself. • The polymerase extends the 3'-OH end and later the loop at 3' end is opened by the scissoring action of S1 nuclease. Restrictio endonucleases and DNA ligase are then used to clone the sequences into bacterial plasmids. • The cloned bacteria are then selected, commonly through the use of antibiotic selection. Once selected, stocks of the bacteria are created which can later be grown and sequenced to compile the cDNA library.
  13. 13. Screening  Once a particular DNA fragment is identified, it can be isolated and amplified to determine its sequence. If we know the partial sequence of a gene and want to determine its entire sequence, the probe should contain the known sequence so that the detected DNA fragment may contain the gene of interest.
  14. 14. cDNA library vs. Genomic Library • In Eukaryotic genomes, the entire sequence of genes is often very large because coding sequence (exons) is interrupted by introns that do not contain coding DNA sequence. • The advantage of a cDNA library is that only the exons, or DNA representing expressed sequence, are templates for the creation of DNA (via Reverse Transcriptase) to collect the preferred genes. • Prokaryotes generally do not contain the apparatus necessary to splice together exons. Furthermore, the introns can increase the genomic space taken up by the gene to a point where prokaryotic genetic manipulation schemes cannot handle the length of the DNA.
  15. 15. • When generating gene libraries from prokaryotic species, Genomic DNA libraries are often used. Genes from prokaryotic cells generally do not contain introns, and the DNA is therefore much shorter, and the RNA does not require much post- processing to be expressed correctly. • Also, prokaryotic mRNA can be difficult to isolate; because of this a genomic library is more preferable for prokaryotic genomic studies. • Therefore both Genomic and cDNA libraries are important in their own aspects and hold key to the various unanswered questions in context to the book of life- the Genome.
  16. 16. Digestion of the chromosomal DNA with restriction endonuclease Production of cDNA (complimentary DNA) Genomic Library Insertion of each DNA fragment into vector (recombinant DNA) Transformation of Bacteria using the recombinant vectors Cloning of the bacterial cells Each clone produced is a “book” in the “Library” of DNA fragments cDNA Library Extraction of mRNAExtraction of chromosomal DNA Insertion of each cDNA into vector (recombinant DNA) Transformation of bacteria Cloning of each recombinant Production of Bacterial cell (clones) containing the gene of interest
  17. 17. cDNA Library uses  cDNA libraries are commonly used when reproducing eukaryotic genomes, as the amount of information is reduced to remove the large numbers of non-coding regions from the library.  cDNA libraries are used to express eukaryotic genes in prokaryotes.  Prokaryotes do not have introns in their DNA and therefore do not possess any enzymes that can cut it out during transcription process. cDNA does not have introns and therefore can be expressed in prokaryotic cells. cDNA libraries are most useful in reverse genetics where the additional genomic information is of less use.  Also, it is useful for subsequently isolating the gene that codes for that mRNA.
  18. 18. Comparison between the library construction

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