Finding Genes

In our consideration of gene cloning, we've glossed over a problem of major significance: How do we find the DNA sequence to be cloned in the first place? In fact, this problem is frequently the most significant one in cloning, because there are often millions or billions of base pairs of DNA in a cell. A discussion of how to solve this problem has been purposely delayed until now because, paradoxically, one must often clone a gene to find it.

This approach—to clone first and search later—is called "shotgun cloning," because it is like hunting with a shotgun: one sprays one's shots widely in the general direction of the quarry, knowing that there is a good chance that one or more of the pellets will hit the intended target. In shotgun cloning, one first clones a large number of DNA fragments, knowing that one or more contains the DNA of interest, and then searches for the fragment of interest among the clones.

A collection of clones containing all the DNA fragments from one source is called a DNA library. For example, we might isolate genomic DNA from human cells, break it into fragments, and clone all of them in bacterial cells or phages. The set of bacterial colonies or phages containing these fragments is a human genomic library, containing all the DNA sequences found in the human genome.

Creating a genomic library To create a genomic library, cells are collected and disrupted, which causes them to release their DNA and other cellular contents into an aqueous solution. There are several methods for isolating the DNA from the other cellular contents. In one method, phenol (an organic solvent that does not mix well with water) is added to the mixture, which is then shaken. The proteins from the cell associate with phenol, whereas the DNA and RNA remain in the aqueous solution, which is removed with the use of a pipette. The nucleic acids are then precipitated from this so lution by adding cold alcohol. RNA can be removed by adding an enzyme that degrades RNA but not DNA.

When DNA has been extracted, it is cut into fragments by using a restriction enzyme to digest it for a limited amount of time only (a partial digestion) so that only some of the restriction sites in each DNA molecule are cut. Because which sites are cut is random, different DNA molecules will be cut in different places, and a set of overlapping fragments will be produced (IFigure 18.13). The fragments are then joined to plasmid, phage, or cosmid vectors, which can be transferred to bacteria. This technique produces a set of bacterial cells or phage particles containing the overlapping genomic fragments. A few of the clones contain the entire gene of interest, a few contain parts of the gene, but most contain fragments that have no part of the gene of interest.

A genomic library must contain a large number of clones to ensure that all DNA sequences in the genome are represented in the library. A library of the human genome formed by using cosmids, each carrying a random DNA fragment from 35,000 to 44,000 bp long, would require about 350,000 cosmid clones to provide a 99% chance that every sequence is included in the library.

Creating a cDNA library An alternative to creating a genomic library is to create a library consisting only of those DNA sequences that are transcribed into mRNA (called a cDNA library because all the DNA in this library is complementary to mRNA). Much of eukaryotic DNA consists of repetitive (and other DNA) sequences that are not transcribed into mRNA (see p. 000 in Chapter 11), and the sequences are not represented in a cDNA library.

A cDNA library has two additional advantages. First, it is enriched with fragments from actively transcribed genes. Second, introns do not interrupt the cloned sequences; introns would pose a problem when the goal is to produce a eukaryotic protein in bacteria, because most bacteria have no means of removing the introns.

The disadvantage of a cDNA library is that it contains only sequences that are present in mature mRNA. Introns and any other sequences that are altered after transcription are not present; sequences, such as promoters and enhancers, that are not transcribed into RNA also are not present in a cDNA library. It is also important to note that the cDNA library represents only those gene sequences expressed in the tissue from which the RNA was isolated. Furthermore, the frequency of a particular DNA sequence in a cDNA library depends on the abundance of the corresponding mRNA in the given tissue. In contrast, almost all genes are present at the same frequency in a genomic DNA library.

To create a cDNA library, messenger RNA must first be separated from other types of cellular RNA (tRNA, rRNA, snRNA, etc.). Most eukaryotic mRNAs possess a string of adenine nucleotides at the 3' end, and this poly(A) tail provides a convenient hook for separating eu-karyotic mRNA from the other types. Total cellular RNA is isolated from cells and poured through a column packed

Special cloning vectors are used for introducing genes into eukaryotes; they include shuttle vectors, which can reproduce in two different hosts, yeast and bacterial artificial chromosomes, which hold DNA fragments hundreds of thousands of base pairs in length, and the Ti plasmid, which transfers genes to plants.

^ Multiple copies of genomic DNA are digested by a restriction enzyme for a limited time so that only some of the restriction sites in each molecule are cut.

Genomic DNA

0 0

Post a comment