Genomic DNA & cDNA Genomic DNA & cDNA LibrariesLibraries

By: Tal H HassonBy: Tal H Hasson

IntroductionIntroduction

The use of genetic information is a The use of genetic information is a powerful tool that today is becoming more powerful tool that today is becoming more readily available to scientists.readily available to scientists.In order to use this powerful tool it In order to use this powerful tool it necessary to know how to navigate necessary to know how to navigate throughout the entire genome. The throughout the entire genome. The human genome is about 3 x 10E9 bp.human genome is about 3 x 10E9 bp.In humans this project is known as Human In humans this project is known as Human Genome Project.Genome Project.

How to generate Genomic How to generate Genomic informationinformation

There two ways in which There two ways in which genomic information is genomic information is obtained.obtained.

Genomic library which Genomic library which contains the entire human contains the entire human Genome (exons and Genome (exons and introns)introns)

cDNA (complementary cDNA (complementary DNA) library the contains DNA) library the contains only expressed genomic only expressed genomic information (only exons)information (only exons)

λλ-phage as a Vector-phage as a Vector

The genomic library is The genomic library is generated by using generated by using λλ-phage -phage for the following reasons.for the following reasons.

1.1. A large number of A large number of λλ phage can be phage can be screened simultaneously (screened simultaneously (5 x 10E4 5 x 10E4 phage plagues).phage plagues).

2.2. λλ phage as a higher transformation phage as a higher transformation efficiency about 1000 times higher efficiency about 1000 times higher compared to a plasmid.compared to a plasmid.

The vector as to maintain its The vector as to maintain its lytic growth.lytic growth.Lysogenic pathway and Lysogenic pathway and other viral genes that are not other viral genes that are not important are replaced with important are replaced with the DNA to be cloned. the DNA to be cloned.

λλ-phage as a Vector -phage as a Vector (Cont.)(Cont.)

An infected E.Coli will produce An infected E.Coli will produce what are know as what are know as concatomersconcatomers (which is the viral genome) on (which is the viral genome) on either site of the either site of the concatomersconcatomers there is a site called COS Site.there is a site called COS Site.Two proteins recognize this site A Two proteins recognize this site A protein and Nu protein, which will protein and Nu protein, which will lead to the insertion of the lead to the insertion of the λλ DNA DNA into the phage head. The into the phage head. The chromosomal DNA that lacks the chromosomal DNA that lacks the COS sites will not enter the phage COS sites will not enter the phage head. Once the genetic head. Once the genetic information is inserted the tail will information is inserted the tail will assemble.assemble.A 50kb can be inserted into the A 50kb can be inserted into the phage.phage.

Generating A Genomic LibraryGenerating A Genomic Libraryλλ-phage is treated with restriction -phage is treated with restriction enzymes that produce enzymes that produce λλ arms with arms with sticky end. These arms contain all sticky end. These arms contain all the lytic genetic information that is the lytic genetic information that is needed for replication and needed for replication and produces room for insertion of new produces room for insertion of new genetic information.genetic information.DNA sequence is obtain from the DNA sequence is obtain from the cell of interest. It is cleaved with cell of interest. It is cleaved with restriction enzymes that produce restriction enzymes that produce 20kb fragments that have 20kb fragments that have complementary sticky ends.complementary sticky ends.Both are mixed in equal amounts Both are mixed in equal amounts and are treated with a DNA ligase and are treated with a DNA ligase that cleaves them together.that cleaves them together.Afterward the entire combined Afterward the entire combined sequence is packed to the phage sequence is packed to the phage head.head.

Packaging of the Recombinant Packaging of the Recombinant DNADNA

To prepare the phage an E.coli cell is infected with a mutant To prepare the phage an E.coli cell is infected with a mutant λλ-phage that as -phage that as a defective “A-protein” (which is one of two genes that are responsible for a defective “A-protein” (which is one of two genes that are responsible for packaging genetic information).packaging genetic information).Therefore the E.Coli accumulates empty heads and also preassembled Therefore the E.Coli accumulates empty heads and also preassembled tails.tails.Once enough heads and tails are assembled we lysate the E.Coli cells.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 To the mixture of heads and tail we add isolated A protein (obtained from E.Coli infected with E.Coli infected with λλ-phage). -phage). In the next step we add the recombinant DNA that has the In the next step we add the recombinant DNA that has the λλ-phage genetic -phage genetic information (which also includes COS sites). information (which also includes COS sites). At this point we have a mixture containing mutant At this point we have a mixture containing mutant λλ-phage heads and tails. -phage heads and tails. There is isolated A protein and recombinant DNA containing There is isolated A protein and recombinant DNA containing λλ-phage -phage genetic information with COS sites. genetic information with COS sites. Therefore we have all the components necessary to package the Therefore we have all the components necessary to package the recombinant DNA into the recombinant DNA into the λλ-phage head. Once the information is inserted -phage head. Once the information is inserted the tail assembles and we have an infectious phage that contains the the tail assembles and we have an infectious phage that contains the recombinant DNA sequence.recombinant DNA sequence.

Generating A Genomic Library Generating A Genomic Library (cont.)(cont.)

In order to sequence the entire In order to sequence the entire genome it is necessary to produce genome it is necessary to produce overlapping sequences. overlapping sequences. Using a technique called chromosome Using a technique called chromosome walking, it is possible to order to walking, it is possible to order to genomic clones.genomic clones.As I mentioned earlier the human As I mentioned earlier the human genome contains 3 × 10E9 base pairs. genome contains 3 × 10E9 base pairs. Each vector contains 20kb that means Each vector contains 20kb that means that it is necessary to generate about that it is necessary to generate about 1.5 x 10E5 phages.1.5 x 10E5 phages.You can screen 5 × 10E4 plaques on You can screen 5 × 10E4 plaques on each petri dish meaning that you can each petri dish meaning that you can contain all the human genome on 20-contain all the human genome on 20-30 petri dishes.30 petri dishes.If plasmids were used instead of If plasmids were used instead of λλ phage it would take 5000 petri dishes.phage it would take 5000 petri dishes.

Generating A cDNA LibraryGenerating A cDNA Library

All eukaryotes have an mRNA. Each All eukaryotes have an mRNA. Each specialized cell have a specific mRNA that specialized cell have a specific mRNA that encodes information for a specific protein.encodes information for a specific protein.

This information can be transformed back into This information can be transformed back into DNA or in other words a DNA copy of mRNA DNA or in other words a DNA copy of mRNA (using reverse transcriptase). This cDNA can be (using reverse transcriptase). This cDNA can be stored in plasmids or phages.stored in plasmids or phages.

cDNA contains only the expressed genetic cDNA contains only the expressed genetic information which allows us to study the amino information which allows us to study the amino acid sequence directly from the DNA.acid sequence directly from the DNA.

Generating A cDNA LibraryGenerating A cDNA Library(Cont.)(Cont.)

The first step in creating a The first step in creating a cDNA library is to isolate cDNA library is to isolate mRNA from the cell. mRNA from the cell.

All mRNA have a poly A All mRNA have a poly A tail (unlike tRNA and tail (unlike tRNA and rRNA that don’t). By rRNA that don’t). By using a column that using a column that contains a short poly T contains a short poly T sequence it is possible to sequence it is possible to isolate the mRNA for both isolate the mRNA for both tRNA and rRNA.tRNA and rRNA.

Generating A cDNA LibraryGenerating A cDNA Library(Cont.)(Cont.)

Once the mRNA is isolated it is treated Once the mRNA is isolated it is treated with an enzyme reverse transcriptase with an enzyme reverse transcriptase (which is found in retro viruses like HIV). (which is found in retro viruses like HIV). This enzyme will create a (ss)cDNA This enzyme will create a (ss)cDNA intermediate from the mRNA.intermediate from the mRNA. By hybridizing the poly A of the mRNA By hybridizing the poly A of the mRNA with oligo T’d a primer is created. with oligo T’d a primer is created. Reverse transcriptase recognizes this Reverse transcriptase recognizes this template and will add bases to 3’ end.template and will add bases to 3’ end.

Generating A cDNA LibraryGenerating A cDNA Library(Cont.)(Cont.)

At this point the (ss)cDNA needs At this point the (ss)cDNA needs to be converted to the double to be converted to the double strand cDNA.strand cDNA.The mRNA cDNA complex is The mRNA cDNA complex is treated with an alkali which treated with an alkali which hydrolyzes the mRNA, but not the hydrolyzes the mRNA, but not the cDNA.cDNA.Then by using Then by using terminal transferaseterminal transferase which is a DNA polymerase that which is a DNA polymerase that adds deoxynucleotides to free 3 adds deoxynucleotides to free 3 ends without the need of template ends without the need of template (this will add poly G).(this will add poly G).To this a synthetic poly C is To this a synthetic poly C is hybridized which is used as primer hybridized which is used as primer for the synthesis of the for the synthesis of the complementary strand of the complementary strand of the cDNA.cDNA.

Packing the cDNAPacking the cDNAThe first step is to ligate to each The first step is to ligate to each end of the cDNA a short end of the cDNA a short restriction-site linkersrestriction-site linkers (which are (which are prepared by bactiophage T4). prepared by bactiophage T4). This will produce blunt end at the This will produce blunt end at the end of the DNA.end of the DNA.Now it is necessary to protect the Now it is necessary to protect the cDNA from unwanted digestion by cDNA from unwanted digestion by restriction enzymes. Therefore restriction enzymes. Therefore the cDNA is treated with a the cDNA is treated with a modification enzyme that modification enzyme that methylates specific bases within methylates specific bases within the restriction enzyme sequence.the restriction enzyme sequence.The next step is to treat the cDNA The next step is to treat the cDNA with restriction enzymes that are with restriction enzymes that are specific to the blunt ends. This will specific to the blunt ends. This will result with sticky end.result with sticky end.

Packing the cDNAPacking the cDNA(cont.)(cont.)

The final step is to The final step is to ligate the sticky ends ligate the sticky ends of the cDNA with the of the cDNA with the λλ-phage arms that -phage arms that have complementary have complementary sticky ends, thereby sticky ends, thereby inserting the Double inserting the Double strand cDNA into the strand cDNA into the vector.vector.

ReferencesReferences

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowSection&rid=mcb.section.1611

Molecular Cloning: A Laboratory ManualJoseph Sambrook, David W. Russell