The Unusual Origin of the Polymerase Chain Reaction
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.. The Unusual Origin of the Polymerase Chain Reaction A surprisingly simple method for making unlimited copies of DNA fragments was conceived under unlikely circumstances-during a moonlit drive through the mountains of California by Kary B. Mullis ometimes a good idea comes tal tissue specimen, from a single hu- it at random points along its length. to you when you are not looking man hair, from a drop of dried blood Consequently, if the DNAis removed S for it. Through an improbable at the scene of a crime, from the tis- from 1,000 identical cells, there will combination of coincidences, naivete sues of a mummified brain or from a be 1,000 copies of any given gene, but and lucky mistakes, such a revelation 40,000-year-old woolly mammoth fro- each copy will be on a DNAfragment came to me one Friday night in April, zen in a glacier. of differing length. 1983, as I gripped the steering wheel In the seven years since that night, For years this problem made it diffi- of my car and snaked along a moonlit applications for the PCRhave spread cult to study genes. Then in the 1970's mountain road into northern Califor- throughout the biological sciences: enzymes known as restriction endo- nia's redwood country. That was how I more than 1,000 reports of its use nucleases were discovered: these en- stumbled across a process that could have been published. Giventhe impact zymes snipped strands of DNAat spe- make unlimited numbers of copies of of the PCRon biological research and cific points. The endonucleases made genes, a process now known as the its conceptual simplicity, the fact that it possible to cut DNA into smaller, polymerase chain reaction (PCR). it lay unrecognized for more than 15 sturdier, more identifiable pieces and Beginningwith a single molecule of years after all the elements for its thereby made it easier to isolate the the genetic material DNA,the PCRcan implementation were available strikes pieces containing a gene of interest. generate 100 billion similar molecules many observers as uncanny. By the late 1970's, therefore, mo- in an afternoon. The reaction is easy to lecular biologists were busily study- execute: it requires no more than a he polymerase chain reaction ing DNAwith endonucleases and with test tube, a few simple reagents and a makes life much easier for mo- other molecules called oligonucleotide source of heat. The DNAsample that Tlecular biologists: it gives them probes. An oligonucleotide is a short one wishes to copy can be pure, or it as much of a particular DNAas they chain of specifically ordered nucle- can be a minute part of an extremely want. Casual discussions of DNAmol- otide bases. Under the right condi- complex mixture of biological materi- ecules sometimes make them sound tions, an oligonucleotide willbind spe- als. The DNAmay come from a hospi- like easily obtained objects. The truth cifically with a complementary -se- is that in practice it is difficult to get a quence of nucleotides in single-strand well-defined molecule of natural DNA DNA.Therefore, radioactively labeled, KARYB.MUlliSdescribes himself as from any organism except extremely man-made oligonucleotides can serve "a generalist with a chemicalprejudice." simple viruses. as probes for determining whether a In addition to the polymerase chain re- action, he is also known for having in- The difficulty resides in the nature sample of DNAcontains a specific nu- vented a plastic that changes color rap- of the molecule. DNA is a delicate cleotide sequence or gene. In 1979 the idly when exposed to ultraviolet light. chain made of four deoxynucleotides: Cetus Corporation in Emeryville,Calif., Whileworking as a biochemistry grad- deoxyadenylate (A),deoxythymidylate hired me to synthesize oligonucleo- uate student at the University of Cali- (T), deoxyguanylate (G) and deoxycy- tide probes. fornia, Berkeley,he published a paper tidylate (C);the sequence of these bas- By 1983 the charm of synthesizing in Nature entitled "The Cosmological es encodes the genetic information. oligonucleotides for a living had en- Significance of Time Reversal." Mullis tered a decline-a decline that most received his Ph.D. in biochemistry in Rarelydoes one find a single strand of r 1972. After working as a postdoctoral DNA;. usually pairs of strands with of us so employed were happy to wit- fellowat the Universityof Kansas Medi- complementary sequences form dou- ness. The laborious but very quaint cal Schooland the Universityof Califor- ble helixes in which the A's in one chemical art form for making oligonu- I nia, San Francisco,Mullisjoined the Ce- strand bind with the T's in.the other, cleotides manually, to which we had ~ tus Corporation, where he discovered and the G's bind with the C's [see grown comfortably numb, had given , I the polymerase chain reaction. In 1986 illustration on opposite page]. Inside way to a much less charming but reli- he became the director of molecular a cell this DNA helix is surrounded able automated technique. It was an biology at Xytronyx, Inc. Today Mullis I and further coiled by various proteins. immense improvement. works in Lajolla, Calif.,as a private con- In the aftermath of this minor sultant on polymerase-chain-reaction When biologists try to isolate a naked technology and nucleic acid chemistry. DNAchain, the DNAis so long and thin industrial revolution, we nucleotide that even mild shearing forces break chemists found ourselves success- 56 SCIENTIFICAMERICAN April 1990 fully underemployed. Laboratory ma- facts about DNA.A strand of the mole- if the template nucleotide is a G, the Q chines, which we loaded and watched, cule has one end that is known, by enzyme attaches a C.Byrepeating this were making almost more oligonucle- chemical convention, as three-prime process, the polymerase can extend otides than we had room for in the and one end that is five-prime. In a the primer's three-prime end all the freezer and certainly more than the double helix of DNA,the complemen- way to the template's five-prime ter- molecular biologists-who seemed to tary strands are said to be antiparallel, minus [see illustration on page 59]. In be working even more slowlyand tedi- because the three-prime end of one a double helix of DNA, each strand ously than we had previously suspect- strand pairs with the five-prime of the serves as a template for the other II ed-could use in their experiments. other strand, and vice versa. during replication and repair. Consequently, in my laboratory at Ce- In 1955Arthur Kornberg of Stanford Now for dideoxy sequencing, which I tus, there was a fair amount of time University and his associates discov- is also commonly called the Sanger i available to think and to putter. ered a cellular enzyme called a DNA technique after one of its inventors, I found myself puttering around polymerase. DNA polymerases serve Frederick Sanger of the British Medi- II Ii, with oligonucleotides. several natural functions, including cal Research CouncilLaboratory of Mo- :1 the repair and replication of DNA. lecular Biology.This technique uses knew that a technique for easily These enzymes can lengthen a short a DNApolymerase, template strands, determining the identity of the nu- oligonucleotide "primer" by attaching primers, nucleotide triphosphates and Icleotide at a given position in a an additional nucleotide to its three- special dideoxynucleotide triphos- DNAmolecule would be useful, espe- prime end, but only if the primer is phates (ddNTP's) to determine DNA cially if it would work when the com- hybridized, or bound, to a comple- sequences. like ordinary nucleotides, plexity of the DNAwas high (as it is in mentary strand. called the template. ddNTP's can be attached to growing human DNA)and when the available The surrounding solution must also primers by polymerases; however, a quantity of the DNAwas small. I did contain nucleotide triphosphate mol- ddNTPwill "cap" the three-prime end not see why one could not use the ecules as building blocks. of a primer and prevent the addition enzyme DNApolymerase and a varia- The nucleotide that the polymerase of any more bases. The Sanger tech- tion of a technique called dideoxy se- attaches willbe complementary to the nique produces primers that have quencing, and so I designed a simple- base in the corresponding position on been lengthened to varying extents minded experiment to test the idea. the template strand. For example, if and then capped by a ddNTP.By ar- To understand the approach I had the adjacent template nucleotide is an ranging these fragments according to in mind, it is worth reviewing certain A, the polymerase attaches a T base; length and by knowing which ddNTP's --~-- 'II' ~ -"".. 110 II DNAconsists of two strands of linked nucleotides: deoxyaden- are always opposite T's, and the G's are opposite C's-and this ylates (A's), deoxythymidylates (T's), deoxyguanylates (G's) complementarity binds the strands together. Each strand has a and deoxycytidylates (C's). The sequence ofnucleotides in one three-prime and a five-prime end Because their orientations strand is complementary to that in the other strand-the A's oppose one another, the strands are said to be antiparallel. SCIENTIFICAMERICAN April 1990 57 ... have been added, an investigator can the presence of a C in the template. would therefore terminate immediate- determine the sequence of bases in In the modified version of this ly after the addition of one base from the template strand. For example, if technique that I was contemplating, a ddNTP to the chain. If I knew which a dideoxyadenine (ddA) base were I would use" only polymerases, tem- ddNTP had been added to the primers, added at a given position, the corre- plates, ddNTP's and primer mole- I would also know the identity of the sponding complementary base in the cules-that is, I would omit the ordi- corresponding base in the template template would be a T; the addition nary nucleotide triphosphates from strand.