Recombinant Clone for Mapping and Sequencing 11 Larry L
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recombinant clone for mapping and sequencing 11 Larry L. Deaven ntil the 1970s it was nearly impossible to isolate and purify single genes in sufficient quantity for biochemicalu analysis and DNA-sequence determination. The difficulty was largely due to the small size of many genes (2000 to 10,000 base pairs, or 2 to 10 kbp) and the large size of complex genomes such as the human genome (3 billion base pairs). In order to obtain 1 milligram of a 2-kbp human gene, such as the ~-globin gene, all of the DNA in all of the cells of twenty-four people would have to be used as the starting material. Even if it were practical to obtain that much DNA, the problem of separating the DNA sequences that encode ~-globin from the rest of the DNA would be very difficult. A solution to thk problem was found during the recomblnant- DNA revolution through the development of a technique called molecula cloning. By using moleculw-cloning techniques, a small fi-agment of DNA can be duplicated, or amplified, into an unlimited number of copies. Shown on these pages are the two common host cells for molecular cloning, the bacterium E. coli and the yeast S. cerevisiae; a popular cloning vector, the Aphage, with its icosahedral head and long tail; a membrane containing a gridded array of recombinant clones to which DNA probes have been hybridized; and a robotic device developed at the Luboratwy that creates those gridded arrays. 219 DNA Libraries Molecular cloning of a gene requires trillions of copies, of the /3-globin gene. to replace any portions of the library three ingredients: one copy or a few Molecular cloning removed the barriers that have been consumed. Therefore, a copies of the gene to be cloned, a that had prevented the biochemical and library is, in a sense, permanent. It can biological cloning vector, and a host cell. molecular analysis of individual genes be repeatedly used or shared with other Cloning vectors are small molecules in complex genomes. laboratories with little or no depletion of of DNA, often circular, that can be During the recombinant-DNA revo- the original recombinant clones. replicated within a host cell. Host cells lution of the 1970s molecular cloning Just as there are legal libraries and are usually single-celled organisms such was also applied to the study of entire medical libraries and scientific libraries, as bacteria and yeast. The first step genomes with even more dramatic there are various types of DNA libraries. of the cloning process is to combine results. In that application, instead Each type is classified according to the the DNA fragment containing the gene of cloning one gene at a time, all vector used in library construction and sequence with the DNA of the cloning of the DNA in a genome is cut into the source of insert DNA. For example, vector. If the vector DNA is circular, the small fragments and each of those DNA libraries constructed from the circle is cut and the gene to be cloned is fragments is cloned. The resulting DNA in human cells are called human- joined to each end of the opened circle, collection of cloned fragments is called genomic libraries. Ideally they contain The new, somewhat larger circle of a DNA library. The word “library” all of the DNA sequences present in DNA is called a recombinant molecule, was chosen because collectively, those the human genome. Human cDNA as is any molecule formed from a cloned fragments contain all of the libraries contain only those sequences cloning vector and an inserted DNA genetic information in an organism. utilized in protein coding. They are fragment. The recombinant molecule Like a library of reference books, a constructed by isolating messenger RNA can now be allowed to enter a host cell, library of cloned human DNA, for (mRNA) molecules from human tissue where it is duplicated by the replication example, represents a collection of and converting them into complementary machinery of the host cell. Each time reference material for studying the DNA (cDNA) by the action of the en- the recombinant molecule is replicated genetic information in human beings. zyme reverse transcriptase. The cDNA a new copy of the gene it contains is However, whereas conventional libraries fragments are then cloned. Because produced. Furthermore, each of the two are ordered collections of information, mRNA molecules are derived from the daughter cells’ formed by the division DNA libraries are unordered and un- protein-coding portions of genes (see of the original host cell receives copies characterized collections of recombinant “Protein Synthesis” in “Understanding of the recombinant molecule. When the clones. Those collections provide the Inheritance”), cDNA libraries contain host cell has grown into a colony, it starting materials for almost all the the sequences within genes that are is referred to as a recombinant clone, current techniques used to decipher the expressed as proteins. Thus a brain and the DNA fragment contained within instructions contained in DNA. cDNA library would be made from the each cell of the colony is said to have Two general features of libraries make mRNAs in brain cells and would contain been cloned. them a remarkable resource. First, only those DNA sequences expressed If we apply the cloning process to the individual clones from a library can as brain proteins. Similarly, a liver production of 1 milligram of the human easily be isolated from the other clones. cDNA library would contain those DNA $-globin gene, a few copies of the gene If the host cells are bacteria, a small sequences whose expression as protein would be inserted into plasmid cloning portion of the library can be placed on is necessary to the proper functioning vectors. (Plasmids are small circular a culture dish where each bacterium of liver cells. DNA molecules found in bacteria.) will form a colony of identical cells. A library is further classified accord- The recombinant plasmids would then Each colony can then be transferred to ing to the vector used in its construction. be added to E. coli bacterial cells. an individual culture dish and grown Since different vectors tend to carry Some of the cells would be entered, or into a large population. Since each DNA inserts with a limited range of “transformed,” by a recombinant plasmid population contains a different cloned lengths, classification by cloning vector, and would begin to produce copies DNA insert, any region of the genome in effect, specifies the average length of the cloned /3-globin gene. Using can be made accessible for analysis and of the inserts within the recombinant this approach, just 2 liters of nutrient sequencing. Second, a DNA library is clones of the library. Each type of solution would produce enough E. coli a renewable resource. The clones can library offers particular advantages for cells to yield 1 milligram, or many be grown individually or collectively particular applications. 220 Los Alamos Science Number 20 1992 DNA Libraries A primary goal of the Human Genome Project is to construct a physical map of ECORI restriction sites J + each human chromosome. A physical ————— 5 GIAATTC _—— —_ GflAATTC map of a chromosome is an ordered CT TAA[G ————— CT TAA[G s collection of clones selected from one or more DNA libraries. Collectively, Cleavage by ECORI endonuclease those clones carry inserts that include Single- stranded \ I all of the DNA in the chromosome, and through the mapping process each cloned insert is ordered according to “;ky’’end 1 its position along the length of the 5;!?<$<<=: c T 7A A ~, —Singje. chromosome (see “Physical Mapping” restriction EcoRI stranded in “Mapping the Genome”). Thus fragment “sticky” end the construction of a physical map is somewhat analogous to the cataloging Figure 1. Restriction-Enzyme Cleavage of documents in a conventional library. A restriction enzyme cleaves DNA at each recognition site on the DNA molecule. In Many of the recent improvements in this illustration the restriction enzyme is EcoRI, which cleaves DNA having the sequence cloning technology have been due to the 5’-GAATTC. Both strands of the DNA are cleaved between the G and A bases, leaving initiation of the Human Genome Project a tail with the sequence TTAA on each cut end. The tails are complementary to each and specifically to the need for physical other, so two pieces of DNA that have both been cleaved with EcoRI can be joined maps of each human chromosome. end-to-end to make a recombinant molecule. If a DNA molecule has tails that facilitate Libraries with large DNA inserts make joining, it is said to have sticky ends. the mapping process both faster and easier, so considerable attention has DNA libraries are vital to much of long series of discoveries and technolog- been given to the development of cloning the research in molecular genetics and ical developments in DNA biochemistry. systems that can faithfully maintain and to most of the activities sponsored by These include the discovery that DNA propagate large DNA inserts. the Human Genome Project, including is the carrier of genetic information in Although much effort is directed to the construction of physical maps, the 1944, the determination that DNA has ordering, or physical mapping, of the sequencing of DNA fragments, the a double-helical structure in 1953, and clones in. a library, unorganized libraries isolation of genes, and the search for the unraveling of the genetic code in are also useful tools. Through a process polymorphic genetic-linkage markers. the 1960s. However, the first essential called library screening, cloned frag- Details of those activities are discussed step in the origin of recombinant-DNA ments of DNA that contain a sequence of elsewhere in this issue.