Connecting Concepts

Cloning Strategies

All gene-cloning experiments have four basic steps:

1. Isolation of a DNA fragment

2. Joining of the fragment to a cloning vector

3. Introduction of the cloning vector, along with the inserted DNA fragment, into host cells

4. Identification of cells containing the recombinant DNA molecule

We've now considered a number of different methods for carrying out these four steps. There is no single procedure for cloning a gene but rather a variety of methods, each with its strengths and weaknesses. The particular combination of methods chosen for a cloning experiment is termed the cloning strategy.

In developing a cloning strategy, a number of factors must be taken into consideration. These factors include how much is known about the gene to be cloned, the size and nature of the gene, and the ultimate purpose of the cloning experiment. The procedure for cloning a small, well-characterized DNA fragment for sequencing would be very different from that for cloning a large, poorly known gene for the commercial production of a protein.

The first step in gene cloning is to find the particular gene or DNA fragment of interest. There are two basic approaches. In one approach, a DNA library can be constructed from genomic or cDNA, and the library can be screened to find the gene of interest. In the other approach, the gene can first be isolated and then cloned. Which approach is used depends largely on what is already known about the gene. Has the gene been mapped? Is there a probe available for screening? Is the amino acid sequence of a protein encoded by the gene known?

If one chooses to make and screen a DNA library, the next problem is to select the best source of DNA. Will it be a genomic library or a cDNA library? If the purpose is to clone the gene in an expression vector and produce a protein, then a cDNA library is ideal. Using a cDNA library means that introns (which bacteria cannot splice out) will be excluded, and fewer colonies will need to be screened. If, on the other hand, the purpose is to examine the regu latory sequences or the introns within a gene, then a genomic library is required.

The next important decision in developing a cloning strategy is to select the cloning vector. The choice depends on a number of factors:

• The length of the sequence to be cloned. For a sequence only a few thousand base pairs in length, a plasmid may be the best choice; if one wants to clone a gene that is 35 kb or longer, a cosmid will be required.

• The organism in which the gene will be cloned. Some vectors are specific for E. coli, whereas others are specific for other bacteria or for eukaryotic cells.

• The selection methods used to find cells containing a plasmid with the inserted gene. One may need a vector with selectable markers so that cells containing the gene can be identified.

• The need for the inserted gene to be expressed. If the protein product is desired, it may be necessary to use an expression vector that contains a promoter and other sequences that ensure transcription and translation of the inserted gene.

• The need for efficiency of transfer to host cells. If selection methods can be used to screen a large number of cells, then a low rate of transfer may be adequate; but, if screening is less efficient or is costly, a higher rate of transfer may be desirable.

A cloning strategy must also take into consideration the best method for joining the DNA fragment and cloning vector. Important points here include the simplicity and ease of the method, the need to retain restriction sites so that the foreign gene can later be retrieved from the vector, and whether the gene sequence must be joined to a promoter and other regulatory sequences to ensure transcription.

The method chosen for moving the vector into the host cell is usually dictated by the type of vector; plasmids are transferred to bacterial cells by transformation, whereas phage vectors and cosmids are transferred by viral infection.

The procedure for screening cells to find those with recombinant molecules depends on how much is known about the cloned fragment, the efficiency of transfer, and the cloning vector used. Considerations used in a developing a cloning strategy are summarized in Table 18.4.

Table 18.4 Considerations in developing a cloning strategy

Step in Gene Cloning

Considerations

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