Библиотеки генетических фрагментов: типы и применение.

Collection of genetic material fragments

In molecular biology, a library is a collection of genetic material fragments that are stored and propagated in a population of microbes through the process of molecular cloning. There are different types of DNA libraries, including cDNA libraries (formed from reverse transcribed RNA), genomic libraries (formed from genomic DNA) and randomized mutant libraries (formed by de novo gene synthesis where alternative nucleotides or codons are incorporated). DNA library technology is a mainstay of current molecular biology, genetic engineering, and protein engineering, and the applications of these libraries depend on the source of the original DNA fragments. There are differences in the cloning vectors and techniques used in library preparation, but in general each DNA fragment is uniquely inserted into a cloning vector and the pool of recombinant DNA molecules is then transferred into a population of bacteria (a Bacterial Artificial Chromosome or BAC library) or yeast such that each organism contains on average one construct (vector + insert). As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated (thus, "cloned").

The term "library" can refer to a population of organisms, each of which carries a DNA molecule inserted into a cloning vector, or alternatively to the collection of all of the cloned vector molecules.

In contrast to the library types described above, a variety of artificial methods exist for making libraries of variant genes. Variation throughout the gene can be introduced randomly by either error prone PCR, DNA shuffling to recombine parts of similar genes together, or transposon based methods to introduce indels. Alternatively, mutations can be targeted to specific codons during de novo synthesis or saturation mutagenesis to construct one or more point mutants of a gene in a controlled way. This results in a mixture of double stranded DNA molecules which represent variants of the original gene. The expressed proteins from these libraries can then be screened for variants which exhibit favorable properties (e. g. stability, binding affinity or enzyme activity). This can be repeated in cycles of creating gene variants and screening the expression products in a directed evolution process.

If creating an mRNA library (i. e. with cDNA clones), there are several possible protocols for isolating full length mRNA. To extract DNA for genomic DNA (also known as gDNA) libraries, a DNA mini prep may be useful.

cDNA libraries require care to ensure that full length clones of mRNA are captured as cDNA (which will later be inserted into vectors). Several protocols have been designed to optimise the synthesis of the 1st cDNA strand and the 2nd cDNA strand for this reason, and also to make directional cloning into the vector more likely. gDNA fragments are generated from the extracted gDNA by using non specific frequent cutter restriction enzymes.

The nucleotide sequences of interest are preserved as inserts to a plasmid or the genome of a bacteriophage that has been used to infect bacterial cells. Vectors are propagated most commonly in bacterial cells, but if using a YAC (Yeast Artificial Chromosome) then yeast cells may be used. Vectors could also be propagated in viruses, but this can be time consuming and tedious. However, the high transfection efficiency achieved by using viruses (often phages) makes them useful for packaging the vector (with the ligated insert) and then introducing them into the bacterial (or yeast) cell. Additionally, for cDNA libraries, a system using the Lambda Zap II phage, ExAssist, and 2 E. coli species has been developed. A Cre Lox system using loxP sites and the in vivo expression of the recombinase enzyme can also be used instead. These are examples of in vivo excision systems. In vitro excision involves subcloning often using traditional restriction enzymes and cloning strategies. In vitro excision can be more time consuming and may require more "hands on" work than in vivo excision systems. In either case, the systems allow the movement of the vector from the phage into a live cell, where the vector can replicate and propagate until the library is to be used.

This involves "screening" for the sequences of interest. There are multiple possible methods to achieve this.