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6. I hovedtræk beskrive hvordan en genbibliotek konstrueres. Angive formålet med et sådan bibliotek. Beskrive hvordan det adskiller sig fra et cDNA-bibliotek (beskrive hvordan mRNA selekteres/mangfoldiggøres og hvordan reverstranskriptase bruges)
Devlin, s.291
Devlin, s.302-4, fig. 7.16

When a complex mixture of thousands of different genes, located on different chromosomes, as in the human genome, is subjected to cleavage by a single restriction endonuclease, thousands of DNA fragments are generated.
 

These DNA fragments are annealed with plasmid vectors that have been cleaved to a linear molecule by the same restriction endonucleases. By adjusting the ratio between the DNA fragments and plasmid vectors, it is possible to incorporate one DNA fragment per plasmid vector.

When the plasmid is incorporated in the bacteria, the bacteria are transformed by the recombinant molecules, such that only one plasmid is taken up by a single bacterium. Each recombinant molecule can be replicated within the bacterium. The bacterium can also divide, still carrying the plasmid vector which multiplies inside the bacteria. The dividing bacterias form colonies.
 

The total bacterial population will contain fragments of DNA that may represent the entire human genome. This is called gene library. A selection system is available to retrieve the gene of interest.

The cloning and amplification of a DNA fragment can be employed in subcloning, mutagenesis and sequences studies. The existence of a gene library makes it possible to access a specific DNA sequence of interest very fast, like looking a book in a library.

cDNA and cDNA-library  

Insertion of specific functional eukaryotic genes into vectors that can be expressed in a prokaryotic cell could produce large amounts of “genetically engineered” proteins with huge medical potential. Unfortunately, it is not possible to clone functional genes from genomic DNA, since eukaryotic genes contain introns that must be spliced out of the primary mRNA transcript. Prokaryotic systems can not splice out the intros, thus the protein will not be functional.

This problem can be avoided by the use of cDNA (complementary DNA) – DNA synthesized from functional (spliced) mRNA by an RNA-dependant DNA polymerase, called reverse transcriptase. The cDNA can then be inserted into a vector, and used for amplification, identification and expression.
 

A cDNA-library is constructed from the total cellular mRNA, by synthesizing cDNA with the help of reverse transcriptase and inserting the cDNA fragments into vectors.

Basic differences between a gene library and cDNA library:

• Gene library contains accidental DNA sequences depending on where the endonuclease has cut, while cDNA contains gene sequences synthesized from a mRNA without any introns
   

• Gene library contains the entire genome, while cDNA library contains the cDNA from a particular cell
   

• It is more difficult to make a cDNA library, because some of the mRNAs are present in small numbers so the mRNA has to be enriched.
   

If only few of interest mRNA copies per cell is present, the mRNA could be difficult to identify and therefore the desired mRNA must be enriched. There are two ways to do that:

1. mRNA can be separated by size by electrophoresis or centrifugation. Knowledge of the molecular weight of the protein encoded by the gene of interest gives a clue to the approximate size of the functional mRNA transcript or its cDNA; variability of the predicted size are due to the non-coding region of the mRNA.
    

2. by using immunological procedures, in which an antibody against the protein are added to the solution, then bind to the growing polypeptide chain. The polypeptide chain is attached to the polysome, from which mRNA can be purified.   

cDNA synthesis

An mRNA mixture is used as a template to synthesize a complementary strand of DNA using a RNA-dependant DNA polymerase, called reverse transcriptase.

A primer is required and advantage is taken of the 3’-end of the mRNA that has a long poly (A) tail to which the primer is aligned. The reverse transcriptase then synthesizes the DNA strand in the presence of the four deoxy ribonucleotide triphosphates.

The cDNA-RNA hybrid is then separated into single-stranded cDNA by melting with heat.  

The 3’-end of the single stranded cDNA molecule makes a hairpin and can be used as a primer in the synthesis of the next cDNA molecule, again by reverse transcriptase.

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