Genomic and cDNA Libraries.ppt

Genomic and cDNA Libraries.ppt

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  Genomic DNA &cDNA Libraries By: Tal H Hasson
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  Introduction The use ofgenetic information is a powerful tool that today is becoming more readily available to scientists. In order to use this powerful tool it necessary to know how to navigate throughout the entire genome. The human genome is about 3 x 10E9 bp. In humans this project is known as Human Genome Project.
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  How to generateGenomic information There two ways in which genomic information is obtained. Genomic library which contains the entire human Genome (exons and introns) cDNA (complementary DNA) library the contains only expressed genomic information (only exons)
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  λ-phage as aVector The genomic library is generated by using λ-phage for the following reasons. 1. A large number of λ phage can be screened simultaneously (5 x 10E4 phage plagues). 2. λ phage as a higher transformation efficiency about 1000 times higher compared to a plasmid. The vector as to maintain its lytic growth. Lysogenic pathway and other viral genes that are not important are replaced with the DNA to be cloned.
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  λ-phage as aVector (Cont.) An infected E.Coli will produce what are know as concatomers (which is the viral genome) on either site of the concatomers there is a site called COS Site. Two proteins recognize this site A protein and Nu protein, which will lead to the insertion of the λ DNA into the phage head. The chromosomal DNA that lacks the COS sites will not enter the phage head. Once the genetic information is inserted the tail will assemble. A 50kb can be inserted into the phage.
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  Generating A GenomicLibrary λ-phage is treated with restriction enzymes that produce λ arms with sticky end. These arms contain all the lytic genetic information that is needed for replication and produces room for insertion of new genetic information. DNA sequence is obtain from the cell of interest. It is cleaved with restriction enzymes that produce 20kb fragments that have complementary sticky ends. Both are mixed in equal amounts and are treated with a DNA ligase that cleaves them together. Afterward the entire combined sequence is packed to the phage head.
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  Packaging of theRecombinant DNA To prepare the phage an E.coli cell is infected with a mutant λ-phage that as a defective “A-protein” (which is one of two genes that are responsible for packaging genetic information). Therefore the E.Coli accumulates empty heads and also preassembled tails. Once enough heads and tails are assembled we lysate the E.Coli cells. To the mixture of heads and tail we add isolated A protein (obtained from E.Coli infected with λ-phage). In the next step we add the recombinant DNA that has the λ-phage genetic information (which also includes COS sites). At this point we have a mixture containing mutant λ-phage heads and tails. There is isolated A protein and recombinant DNA containing λ-phage genetic information with COS sites. Therefore we have all the components necessary to package the recombinant DNA into the λ-phage head. Once the information is inserted the tail assembles and we have an infectious phage that contains the recombinant DNA sequence.
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  Generating A GenomicLibrary (cont.) In order to sequence the entire genome it is necessary to produce overlapping sequences. Using a technique called chromosome walking, it is possible to order to genomic clones. As I mentioned earlier the human genome contains 3 × 10E9 base pairs. Each vector contains 20kb that means that it is necessary to generate about 1.5 x 10E5 phages. You can screen 5 × 10E4 plaques on each petri dish meaning that you can contain all the human genome on 20- 30 petri dishes. If plasmids were used instead of λ phage it would take 5000 petri dishes.
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  Generating A cDNALibrary All eukaryotes have an mRNA. Each specialized cell have a specific mRNA that encodes information for a specific protein. This information can be transformed back into DNA or in other words a DNA copy of mRNA (using reverse transcriptase). This cDNA can be stored in plasmids or phages. cDNA contains only the expressed genetic information which allows us to study the amino acid sequence directly from the DNA.
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  Generating A cDNALibrary(Cont.) The first step in creating a cDNA library is to isolate mRNA from the cell. All mRNA have a poly A tail (unlike tRNA and rRNA that don’t). By using a column that contains a short poly T sequence it is possible to isolate the mRNA for both tRNA and rRNA.
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  Generating A cDNALibrary(Cont.) Once the mRNA is isolated it is treated with an enzyme reverse transcriptase (which is found in retro viruses like HIV). This enzyme will create a (ss)cDNA intermediate from the mRNA. By hybridizing the poly A of the mRNA with oligo T’d a primer is created. Reverse transcriptase recognizes this template and will add bases to 3’ end.
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  Generating A cDNALibrary(Cont.) At this point the (ss)cDNA needs to be converted to the double strand cDNA. The mRNA cDNA complex is treated with an alkali which hydrolyzes the mRNA, but not the cDNA. Then by using terminal transferase which is a DNA polymerase that adds deoxynucleotides to free 3 ends without the need of template (this will add poly G). To this a synthetic poly C is hybridized which is used as primer for the synthesis of the complementary strand of the cDNA.
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  Packing the cDNA Thefirst step is to ligate to each end of the cDNA a short restriction-site linkers (which are prepared by bactiophage T4). This will produce blunt end at the end of the DNA. Now it is necessary to protect the cDNA from unwanted digestion by restriction enzymes. Therefore the cDNA is treated with a modification enzyme that methylates specific bases within the restriction enzyme sequence. The next step is to treat the cDNA with restriction enzymes that are specific to the blunt ends. This will result with sticky end.
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  Packing the cDNA(cont.) Thefinal step is to ligate the sticky ends of the cDNA with the λ-phage arms that have complementary sticky ends, thereby inserting the Double strand cDNA into the vector.
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  References http://www.ncbi.nlm.nih.gov/books/bv.fcgi? call=bv.View..ShowSection&rid=mcb.secti on.1611 Molecular Cloning: ALaboratory Manual Joseph Sambrook, David W. Russell