Sponsored by the U.S. Department of Energy Human Genome Program
Human Genome News Archive Edition
Human Genome News, July 1992; 4(2)
By David L. Nelson
This article is a personal account of events leading to the development of a technique for specifically identifying and amplifying only the human DNA sequences in hybrid cells and in yeast artificial chromosome (YAC), lambda, and cosmid clones. The author describes how insight and a bit of serendipity led to a creative application of the standard polymerase chain reaction (PCR) method, a procedure used to amplify a desired DNA sequence hundreds of millions of times in a matter of hours. Alu PCR, the application devised by Nelson and others, amplifies target sequences falling between segments of DNA that are repeated throughout the human genome. The Alu family of repetitive DNA sequences involves 150- to 300-bp segments containing sequence similarities, including a tetranucleotide that can be cleaved by the restriction enzyme Alu I.Alu repeats constitute nearly 10% of the total human genome.
Alu PCR was originally intended as a technique for identifying human DNA in human-rodent hybrid cells for a proposed reference human genome library. It has proved, however, to be an efficient approach to generating probes from laser microdissected or flow-sorted chromosomal regions. In addition, Alu PCR has facilitated clone ordering based on analysis of PCR patterns, thus making the identification of contiguous clones more efficient; this use has been extended to chromosome walking and expansion of established contigs. The use of PCR primers directed toward repeated DNA sequences has now been extended to include other repeated sequences and combinations of different types of repeated sequences.
-D. Casey, HGMIS
Alu PCR had its origins in January 1987 in Santa Fe, New Mexico, at one of the first meetings ("Exploring the Role of Robotics and Automation in Decoding the Human Genome") on the Human Genome Project. Walter Gilbert, a keynote speaker, proposed something quite provocative: the development of a reference genome on which all investigators would work. This would allow sequence determination of a particular human genome rather than that of a hodgepodge collection of DNA samples produced by the preparation of recombinant libraries and cell lines.
Gilbert also suggested that the best genome for this purpose would be haploid; only one copy of each chromosome (and gene) would be represented, thus eliminating polymorphic variation between chromosomes. He recommended growing large quantities of a haploid tumor (an ovarian teratoma-I don't remember what was to become of the Y chromosome) and distributing the DNA to all genome researchers.
Gilbert's suggestion to begin anew in constructing chromosomal resources was unsettling. Having worked with David Housman [Massachusetts Institute of Technology (MIT)] as a graduate student, I was well schooled in the usefulness of somatic cell hybrid resources and was not ready to shelve them. (Somatic cell hybrids are produced by fusing together human and rodent cells. The preferential loss of human chromosomes during this process results in a hybrid cell with one to a few specific human chromosomes.) Reinventing all the hybrids using a standard cell line to isolate chromosome-specific sequences would be an arduous task.
My thoughts turned instead to the possibility of using the existing hybrids to identify clones in Gilbert's new reference library. I recalled a talk that Kary Mullis had given a month before at Baylor College of Medicine about PCR, which was then a new method on which Mullis had been working at Cetus Corporation. After Mullis's talk many of us spent inordinate amounts of time dreaming of new PCR applications.
This "PCR fever" generated many ideas that became reality at Baylor and elsewhere over the next couple of years. However, it took the trip to Santa Fe and Gilbert's talk to prompt me to consider Alu PCR. The question was simple: Was there a way to amplify only the human DNA in the hybrid cells to produce probes specific for human sequences? Having had extensive experience with the method developed by Housman and Jim Gusella (MIT) using human repeat sequences to identify specific human clones from hybrid cell libraries, I began to wonder whether the Alu repeat (the most common of the short interspersed repeat sequences) could be used to amplify only human sequences. Various schemes came to mind, most of them very convoluted and requiring vector sequence ligation to provide the second primer site for PCR. Amplifying the area between Alu repeats didn't occur to me until later.
On my return to Houston, I began to design primers for amplifying the target sequences by PCR, gathering all the Alu repeats I could identify from the GenBank® sequence database and looking for regions of high homology between copies of human Alu. At first I paid no attention to the rodent Alu-equivalent sequences, assuming that since they did not cross-react in probe hybridization, they would not cross-amplify in PCR. When this assumption turned out to be wrong, I used a region unique to primate sequences that did not amplify rodent equivalents. Because we had no local oligonucleotide facility, I ordered the first Alu primer [designated TC (Tom Caskey)-65] from the central Howard Hughes Medical Institute facility in Boston.
TC-65 arrived in about a month but remained in the freezer for over a year until PCR technology improved enough through the use of Taq polymerase and automated thermal cycling that I was finally ready to try it. Before those improvements, use of the Klenow fragment (a DNA polymerase) and manual shifting of tubes between water baths demanded too much bench bondage. In June 1988 our first try with the new protocol was rewarded-TC-65 provided human-specific amplification in hybrid cells. When most of the next primers I designed did not perform as well (because of cross-amplification with rodent sequences), I realized just how lucky our first efforts had been, since without this initial success I probably would not have pursued further development of the method.
By spring 1989 we had a very nice system for amplifying human sequences from hybrids and had begun to use it for cloned DNA as well. In April I presented a poster on our results at the second Cold Spring Harbor Genome Mapping and Sequencing meeting. To my surprise, Pieter de Jong of Lawrence Livermore National Laboratory presented a poster with very similar results. Later I learned that others had been working out similar primers directed to Alu for the same purpose, including Bryan Young [Imperial Cancer Research Fund (ICRF)] and Paul Goodfellow (University of British Columbia).
Today, use of Alu PCR and its variants has become standard practice for genomicists. Its usefulness as a specific identifier of human sequences extends from hybrid cells to YACs to clones in lambda and cosmid. It is also gratifying to see that the use I had originally envisaged is finally coming to fruition. The ability to use hybrid cell lines to probe "reference" libraries is demonstrated by the work of Tony Monaco (ICRF) and of Daniel Cohen's group at the Centre d'Etude du Polymorphisme Humain and Genethon; these researchers have identified chromosome- and region-specific YAC and cosmid clones by hybridization to Alu PCR products derived from somatic cell hybrids.
So, while Gilbert's idea of ovarian teratomas never caught on, the difficulty involved in producing high-quality YAC libraries (i.e., those having large-insert clones and a small number of chimeric clones) has made necessary the screening of a few "reference" genomes. Dissection of these total human libraries into chromosome- and region-specific sublibraries using somatic cell hybrids and Alu PCR is now a reality, and more interesting applications of PCR (and Alu PCR) are doubtless on the way.
For more information on this topic, refer to
David L. Nelson is Associate Director for the Human Genome Center, Baylor College of Medicine, where his Alu-PCR Research was funded by both DOE and NIH. Director of the Baylor genome center is Thomas Caskey.
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Human Genome Program, U.S. Department of Energy, Human Genome News (v4n2).
The Human Genome Project (HGP) was an international 13-year effort, 1990 to 2003. Primary goals were to discover the complete set of human genes and make them accessible for further biological study, and determine the complete sequence of DNA bases in the human genome. See Timeline for more HGP history.
Published from 1989 until 2002, this newsletter facilitated HGP communication, helped prevent duplication of research effort, and informed persons interested in genome research.