Cloning the DNA

Making the Libraries Cloning the DNA

he cells have been cultured, the each strand: 2 I A as a cloning vector. that is. as a medium chromosomes isolated and sorted. capable of accommodating foreign DNA seg- T Now can begin the final act in the ments for replication. Charon 21A is a modi- making of a library—that of multiplying the DNA in the sorted chromosomes by a factor DNA molecule is about 42,000 base pairs of more than a million. Some tricks of re- long, terminates with short stretches of combinant DNA technology make such great single-stranded DNA, and includes a single amplification possible, and the magicians EcoRI is produced by certain strains of the EcoRI cleavage site.) The DNA molecules who perform these tricks are lowly but ac- bacterium Escherichia coli as a defense extracted from the phages are cut by EcoRI commodating bacteria and viruses. against infection by the DNA of bacterio- into two "arms.” The cut ends of these arms Briefly, four steps are involved in cloning phages. The enzyme severs the viral DNA are then stripped of- a phosphate group (for a the DNA: preparation of DNA fragments and thus prevents its propagation in the reason to be explained later) by treatment from the sorted chromosomes and from bacterium. (To avoid self-destruction, the with the enzyme alkaline phosphatase. bacterial viruses (bacteriophages, or simply bacterium also produces another enzyme The next step is fairly simple, at least in phages); formation of recombinant DNA that modifies its own DNA so that it cannot theory. We mix the two DNA preparations molecules including both chromosomal and be severed by EcoRI.) other bacterial species and let nature take its course—nature in this viral DNA fragments: assembly of phages produce restriction enzymes that recognize case meaning the tendency for single strands containing the recombinant DNA molecules: other sequences. For example, HindIII, the of DNA to “stick together” (become linked and cloning of the phages (and hence replica- restriction enzyme used by our collaborators by hydrogen bonds) at regions with com- tion of the chromosomal DNA fragments) in at Livermore for their phase I libraries, is plementary bases, Since the cutting action of a bacterial host. For concreteness we de- produced by the bacterium Hemophilus in- EcoRI has equipped both the chromosomal scribe, and illustrate in the accompanying fluenzae. Like EcoRI, HindIII recognizes a DNA fragments and the vector arms with figure, the making of what we call a Phase I six-base sequence (–A–A–G-T–T--T–) just such sticky ends. aggregates consisting of’ library, one based on relatively small and makes staggered cuts. two arms linked by a (single) chromosomal chromosomal DNA fragments. (Our Phase 11 Complete digestion of the chromosomal DNA fragment will form. (Herein lies the libraries, which will serve a different DNA with EcoRI (or HindIII) produces a set reason for gentle extraction of the DNA from purpose, will be based on larger fragments. ) of relatively small flagments. We can esti- the chromosomes: physical breaks do not First the DNA molecules in the sorted mate the average length of these fragments result in sticky ends. ) These aggregates arc chromosomes must be extracted from the by assuming a random distribution along the then converted into intact DNA molecules other chromosomal components. For rea- chromosomal DNA of equal numbers of the by treatment with the enzyme DNA ligase, sons that will become clear later, this extrac- four bases A, T, G, and C. Then the prob- which establishes a covalent bond at the tion must be performed very gently to mini- ability of any six successive bases being the break along each strand. The result is a set of mize breaking the very long and very fragile EcoRI (or HindIII) recognition sequence is recombinant DNA molecules—that is, mol- DNA molecules. (1/4)6, which implies an average of about ecules of phage DNA carrying human DNA The extracted DNA is then digested with 4100 base pairs between cleavage sites, (An fragments. (To increase the probability of the restriction enzyme EcoRI, which rec- intact DNA molecule in the largest human forming recombinant aggregates. the ognizes the sequence of six bases chromosome (number 1) is about 2.5 X 108 chromosomal fragments are mixed with an –G–A–A–T–T–C– and, as shown below, base pairs in length. ) excess of arms. Therefore, many of the ag- cuts the DNA at each occurrence of’ this We, and our collaborators at Livermore, gregates formed will consist simply of two sequence by breaking one covalent bond on use DNA from the bacteriopbage Charon arms. But DNA ligase cannot repair the

86 Spring/Summer 1985 LOS ALAMOS SCIENCE Genes by Mail

Extract DNA

III. CLONING THE: DNA

DNA in such aggregates because of the miss- what genes or combinations of genes are ing phosphate groups, So our foresight in the involved; in fact, we haven’t come close to previous step reduces the background of settling even the nature-versus-nurture issue. Digest with EcoRI nonrecombinant DNA molecules in the final And even when a single defective gene is library.) known to be the culprit—as it is in certain Next we assemble Charon 21A phages, kinds of mental retardation—developing the drawing on proteins produced by mutant techniques for introducing the normal gene varieties of the phage for the necessary struc- and getting it expressed properly will take a tural components and on our supply of re- long time. combinant DNA molecules for the genetic HILDEBRAYD: Yes, routine clinical ap- component. This step unavoidably in- plication of gene therapy is a long way off, troduces a deficiency in the resulting library: especially in light of the ethical and legal those recombinant DNA molecules contain- questions it raises, But in the process of Mix and Treat ing fragments longer than about 9100 base ironing out those questions, society should with DNA Ligase pairs are too large to be encased in the heads focus on the extraordinary benefits to be of the phages, and therefore the library will derived from the technology rather than its lack that portion (about 33 mass percent) of “Brave New World” implications. the chromosomal DNA residing on such MOYZIS: I think other developments in fragments. It is for this reason that we and molecular genetics pose more immediate our collaborators at Livermore use different concerns. For instance. the availability of- restriction enzymes and thus produce li- probes specific to the X and Y chromosomes braries based on different sets of chromo- has made it very easy to determine the sex of Assemble Phages somal DNA fragments. The expectation is a fetus. Since even in "sexually liberatcd” that the DNA missing from one laboratory’s western countries the preference for having a I version of a library will be found in the other, male child first is still strong. routine de- The final step in making the libraries, that termination of fetal sex could conceivably of cloning the recombinant DNA molecules, lead to dramatic changes in the ratio of the is accomplished by seeding the phages onto a sexes. The ethical. social, and economic im- “lawn,” or monolayer, of an appropriate plications of this possibility need immediate strain of E. coli, When a phage injects its consideration. Infect Bacteria recombinant DNA molecule into such a cell, SCIENCE: Are issues such as these heavy on the biological apparatus of the cell is your mind as you go about your research? subverted to production of new phages. The CRAM: I don’t think so. The goals of basic cell ruptures when between one and two research arc information and understanding. hundred new phages have been produced, It’s up to society to determine how those are and these then infect surrounding cells. The applied. Take the photoelectric effect, for process continues. resulting in formation of a example. Its applications are mainly viewed plaque, or clear area, in the translucent lawn as beneficial, although a few may be regarded of bacteria. In each plaque arc some one to by some as deplorable. But a scientist doesn’t ten million infective phages, each containing hide his head in the sand in the face of Collect Library of Cloned Phages an exact copy of the recombinant DNA information that can possibly be applied in a molecule carried by the original infecting harmful way. phage. MOYZIS: I would second that. You can’t be These vastly multiplied recombinant paralyzed because a bit of knowledge can be DNA molecules, then, constitute a library- used inappropriately. And 1 repeat that most conveniently and safely packaged within the of the worrisome aspects of gene therapy are proteinaceous armor of phages. This con- a long way down the road. A major problem struction of a library puts us at the starting that must he solved before it can be applied line. Its application to problems of clinical to many diseases is how to introduce the and fundamental significance lies ahead. ■ good genes into only the appropriate CellS of

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