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The following points highlight the top three types of specialized libraries. The types are: 1. Shelves 2. Normalized Libraries 3. Subtractive Libraries.
Specialized Library: Type # 1.
Shelves:
Sometimes we know the size of the restriction fragment on which a particular gene is located. For example, this information may be acquired by probing a Southern blot of digested genomic DNA with a suitable sequence, such as an oligonucleotide probe, and measuring the size(s) of restriction fragment(s) that hybridize.
Once the size of the relevant restriction fragment is known, another digest of genomic DNA is then carried out with the same enzyme. The products are separated by electrophoresis, and DNA fragments of approximately that size are recovered from the gel.
They are then cloned into-a suitable vector. Because they are likely to be smaller than the random fragments used in making full genomic libraries, a plasmid vector is often suitable. The collection of recombinants generated is frequently called a shelf, as it is a subsection of a library.
Specialized Library: Type # 2.
Normalized Libraries:
A library made from cDNA that was prepared directly from mRNA will have a large number of members representing abundant RNAs and few representing the rare RNAs. If the cDNA we are looking for corresponds to an abundant RNA, that will increase the chances of finding an appropriate clone when the library is screened.
However, if the cDNA we are looking for corresponds to a rare RNA, then we will have to screen a large number of members of the library. It is possible to increase the representation of rare mRNAs in a library by a technique called normalization.
In the making of a normalized library a conventional cDNA library is first constructed using the RNaseH method for cDNA generation and cloning into a standard vector. The cDNA inserts are then amplified using primers that flank the cloning site of the vector, melted by heating and then allowed to re-anneal.
Before re-annealing is complete, the DNA is passed down a column of hydroxyapatite, which binds more tightly to double-stranded nucleic acids than to single-stranded nucleic acids.
The single stranded material, therefore, passes through the column. This eluate is enriched for the less abundant sequences (as more abundant sequences will be more likely to have annealed in the time available). It can be enriched again by PCR if need be, and then cloned into a suitable vector as before.
Specialized Library: Type # 3.
Subtractive Libraries:
It is often very useful to make libraries that are enriched for sequences that are present in one sample but which are absent from another. These sequences might be present in the RNA from one tissue type but are absent from the RNA of another. Alternatively, they might be genomic DNA sequences that are present in a wild-type individual but are absent from a mutant that has a deletion in that region of the genome.