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Molecular Biology 1985

Enzymatic Amplification of β-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia

Randall Saiki, Kary Mullis, et al.

Copy one chosen stretch of DNA a billionfold — using nothing but cycles of heat.

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In depth · the introduction

What if you could take one invisible speck of DNA and, in an afternoon, make a billion copies of it — using nothing but a machine that heats and cools?

The big idea

PCR — the polymerase chain reaction — is a way to copy one specific stretch of DNA over and over until there is enough of it to work with. You pick the piece you want by adding two short "bookmarks" called primers, one for each end. Then you run the test tube through a repeating cycle of temperatures.

Each cycle doubles the amount of that piece: 1 becomes 2, 2 becomes 4, 4 becomes 8. That doesn't sound like much — but doubling thirty times turns a single molecule into more than a billion. From a trace too small to see or test, you end up with plenty.

How it came about

The idea is credited to Kary Mullis, a chemist at the Cetus company in California, who said it came to him in 1983 while driving a mountain road at night: copy a piece of DNA, then copy the copies, and let the numbers explode. It was a simple thought, but making it actually work in the lab took a team — and the first published proof, in 1985, came as part of a test for sickle cell anemia, with Randall Saiki as lead author and Mullis among the authors.

Early on it was clumsy. The DNA-copying enzyme they used was killed by the heating step, so a researcher had to open every tube and add fresh enzyme by hand, cycle after cycle. The fix, in 1988, was to borrow an enzyme from a microbe that lives in scalding hot springs — it shrugs off the heat — so the whole thing could be left to run by itself in a machine. Mullis won the 1993 Nobel Prize in Chemistry for the idea; the team around him is why it worked.

Why it mattered

Before PCR, studying a particular gene often meant a slow hunt for enough material. PCR made DNA abundant on demand — and that changed everything downstream. It is why a single hair or a drop of old blood can be matched to a person, why a swab can be tested for a virus, why prenatal and genetic tests are routine, and why reading and editing genomes became practical at all.

A way to picture it

Imagine a photocopier that can only copy one paragraph of a thick book — the paragraph you bracket with two sticky notes. Each pass copies that paragraph, then you feed the copies back in and copy those too. After a handful of passes you're buried in copies of just that paragraph, while the rest of the book is left far behind. PCR is that copier, and the doubling is what makes the pile grow so fast.

An interactive PCR model: a three-step schematic shows one cycle — denature at 94°C splits the double strand, primers anneal at 55°C, and the polymerase extends at 72°C — turning one copy into two. A slider sets the number of cycles and a chart on a logarithmic axis shows the target doubling each round, climbing from one copy through thousands, millions and billions.

Where it sits

PCR works only because Watson and Crick (1953) showed that DNA's two strands are complementary — each strand can rebuild the other, which is exactly what the copying step does. It amplifies the genes that Mendel first inferred as hidden units of heredity. And it is the everyday workhorse behind the newer entries in this Library: the sequencing that feeds AlphaFold, and the edits made by CRISPR, almost always begin with a PCR.

The original document
Original source text
R. K. Saiki, S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich & N. Arnheim · Science 230 (1985): 1350–1354
This paper presents a fast, sensitive prenatal test for sickle cell anemia — and, almost as a tool inside it, the first published description of the polymerase chain reaction. The method that became PCR is the paper's opening move, quoted here from the abstract.
From the abstract
Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia.
The first involves the primer-mediated enzymatic amplification of specific β-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies.
The β-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.
How the amplification works (structural map)
Two short oligonucleotide primers are chosen to flank the β-globin segment of interest. Each cycle of the reaction has three steps: the DNA is heated so the double strand comes apart, cooled so a primer binds to each separated strand, and held warm while a DNA polymerase extends each primer into a full complementary copy. Because every new strand becomes a template in the next cycle, the segment between the two primers roughly doubles each round.
In this 1985 work the polymerase is the Klenow fragment of E. coli DNA polymerase I, which the heating step destroys — so a fresh aliquot of enzyme had to be added by hand at every cycle. Twenty-some cycles raise the chosen target by the ~220,000-fold the abstract reports.
[ … ]
In the second method, the amplified product is examined with an end-labeled oligonucleotide probe and a restriction enzyme: the sickle (βS) mutation alters a restriction site, so the digestion pattern reveals the genotype directly — normal, carrier, or affected — from the enriched DNA.
Cetus Corporation, Emeryville, California · 1985