MoBio DNA Library and Screening Chapter 9

DNA library is a collection of DNA fragments. It may be divided into two types:

The genomic library contains DNA fragments representing the entire genome of an organism.

The cDNA library contains only complementary DNA molecules synthesized from mRNA molecules in a cell.

Preparation of a DNA Library

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Figure 9-B-1. Preparation of the genomic library using λ vectors. It is basically the cloning of all DNA fragments representing the entire genome.

Genomic Library

The genomic library is normally made by λ phage vectors, instead of plasmid vectors, for the following reasons.

The entire human genome is about 3 x 109 bp long whereas a plasmid or λ phage vector may carry up to 20 kb fragment. This would require 1.5 x 105 recombinant plasmids or λ phages. When plating E. coli colonies on a 3" petri dish, the maximum number to allow isolation of individual colonies is about 200 colonies per dish. Thus, at least 700 petri dishes are required to construct a human genomic library. By contrast, as many as 5 x 104 λ phage plagues can be screened on a typical petri dish. This requires only 30 petri dishes to construct a human genomic library. Another advantage of the λ phage vector is that its transformation efficiency is about 1000 times higher than the plasmid vector.

cDNA Library

The advantage of cDNA library is that it contains only the coding region of a genome. To prepare a cDNA library, the first step is to isolate the total mRNA from the cell type of interest. Because eukaryotic mRNAs consist of a poly-A tail, they can easily be separated. Then the enzyme reverse transcriptase is used to synthesize a DNA strand complementary to each mRNA molecule. After the single-stranded DNA molecules are converted into double-stranded DNA molecules by DNA polymerase, they are inserted into vectors and cloned.

Probes

A probe is a piece of DNA or RNA used to detect specific nucleic acid sequences by hybridization (binding of two nucleic acid chains by base pairing) . They are radioactively labeled so that the hybridized nucleic acid can be identified by autoradiography.

The size of probes ranges from a few nucleotides to hundreds of kilobases. Long probes are usually made by cloning. Originally they may be double-stranded, but the working probes must be single-stranded. Short probes (oligonucleotide probes) can be made by chemical synthesis. They are single-stranded.

Suppose we have known the sequence of a specific gene in yeast and want to find its homologous gene in human, then we may use the specific yeast gene as a probe to detect its homologous gene from the human genomic library. On the other hand, if we know the conserved sequence in the specific gene between yeast and human, we may use oligonucleotide probes containing only the conserved sequence. Typically, an oligonucleotide about 20 nucleotides long is sufficient to screen a library.

In some cases, we have known the partial sequence of a protein and want to detect its gene in the library. Then we may synthesize oligonucleotide probes based on the known peptide sequence. Since an amino acid may be encoded by several DNA triplets, many different oligonucleotide probes are often needed.

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Figure 9-B-2. The relationship between a peptide and all possible DNA sequences. In this example, the peptide sequence Leu-Phe-Tyr-Met-His-Asp corresponds to 96 (= 6 x 2 x 2 x 1 x 2 x 2) possible DNA sequences.

Screening

Once a particular DNA fragment is identified, it can be isolated and amplified to determine its sequence. If we know the partial sequence of a gene and want to determine its entire sequence, the probe should contain the known sequence so that the detected DNA fragment may contain the gene of interest.

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Figure 9-B-3. Screening of a specific DNA fragment. After recombinant λ virions form plaques on the lawn of E. coli, the nitrocellulose filter (membrane) is placed on the surface of the petri dish to pick up λ phages from each plaque. Then, the filter is incubated in an alkaline solution to disrupt the virions and release the encapsulated DNA, which is subsequently denatured. Next, the probe is added to hybridize with the target DNA fragment, whose position may be displayed by autoradiography.