15.5: The Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) can amplify a region of DNA from any source, like DNA fragments obtained from a fossil or residue left at a crime scene, or even DNA from a single cell. This amplification usually takes just a few hours, generating millions of copies of the desired target-DNA sequence. The result is the purification of a specific region of DNA from surrounding sequences in a single reaction! Kary B. Mullis was awarded a Nobel Prize in 1993 for his development of PCR, which is now the basis of innumerable research studies of gene structure, function, and evolution as well as applications in criminal forensics, medical diagnostics, and other commercial uses. PCR is described in detail next.

272 PCR: Design and Synthesize Opposing Oligonucleotide Primers

273 PCR: The Amplification Reaction

15.5.1 PCR—the Basic Process

Typical PCR relies on the investigator knowing just two bits of DNA sequence, which will be used to design and to synthesize short oligonucleotide sequences (oligomers) in the laboratory. The oligomers must be complementary to sequences on the opposite strands of doublestranded DNA containing the gene to be studied, with their 3’ ends facing (opposing) each other on either side of the sequence to be amplified. This way, the two oligomers can serve as primers for the replication of both strands of a double-stranded target-DNA sequence. Check out link #272 for further explanation.

The first step in PCR is to add oligomer primers to the target DNA from which a gene (or other genomic sequence) is to be amplified. The mixture is then heated to denature the target DNA. The mixture is cooled to allow the primers to H-bond to complementary targetDNA strands. Next, the four deoxynucleotide precursors to DNA (dATP, dCTP, dTTP and dGTP) are added, along with a small amount of a DNA polymerase. New DNA strands will now lengthen from the oligonucleotide primers on the template DNAs. To make lots of the PCR product, this reaction cycle must be repeated many times. Therefore, after allowing elongation, the mixture is heated to denature (separate) all the DNA strands. When the mixture is again cooled, the oligomers again find complementary sequences with which to Hbond. Early versions of PCR originally relied on an E. coli DNA polymerase, which is inactivated by heating, and so had to be re-added to the PCR mixture for each elongation cycle. When the heat stable Thermus aquaticus DNA polymerase (Taq polymerase) became available, it was adapted for PCR because it is active at high temperatures, eliminating the need to add fresh DNA polymerase after each PCR reaction cycle. It also allowed automation of PCR reactions with programmable thermocyclers, which raised and lowered the temperatures during PCR reactions. PCR amplification with Taq polymerase is shown in Figure 15.27.

Figure 15.27: In PCR, a template DNA to be amplified is mixed with a pair of primers whose sequences target opposite ends of the template. Addition of DNA polymerase (usually Taq Polymerase) initiates PCR. The actual sequence to be amplified (from primer to opposing primer) has been synthesized by the end of the second PCR cycle. Multiple cycles of PCR follow, during which this ‘primer-to-primer’ DNA will be geometrically amplified.

You can see from the illustration that the second cycle of PCR has generated the two DNA strands that will be templates for doubling and redoubling the desired product after each subsequent cycle. A typical PCR reaction might involve thirty PCR cycles, resulting in a nearly exponential amplification of the desired sequence.

CHALLENGE

Starting with a pair of complementary target-DNA molecules after the third PCR cycle, how many double-stranded PCR products should you theoretically have at the end of all thirty PCR cycles?

PCR amplification products are in such abundance that they can be seen under fluorescent illumination on an ethidium bromide-stained agarose gel (Figure 15.28).

Figure 15.28: Agarose-gel electrophoresis of PCR-amplified DNA is stained with ethidium bromide to make the DNA detectable under fluorescent light. PCR products of a globin gene clone from the insect Chironomus thummi are in lanes 2-5. Their sizes (lengths in base pairs) can be determined by comparison to the lengths of bands in a standard-DNA size ladder in lane 1.

Because they are so plentiful, PCR-amplified DNAs can be sequenced and used in many subsequent studies. In this gel, the first lane (on the left) contains a DNA ladder, a mixture of DNAs of known lengths that can be used to estimate the size of the bright bands (i.e., PCR products) in the remaining lanes. In this example, four PCR products were amplified from the same cloned genomic DNA preparation, using different combinations of oligonucleotide primers.

15.5.2 The Many Uses of PCR

PCR-amplified products can be labeled with radioactive or fluorescent tags to screen cDNA or genomic libraries, to determine where a DNA sequence of interest migrates on a Southern blot, or to determine where an RNA sequence of interest migrates on a Northern blot (a fanciful name for RNAs that are separated by size on gels and blotted to filter). In a major PCR advance, Quantitative PCR was developed to study differential gene expression and gene regulation. The technique allows cDNA amplifications from RNAs under conditions that detect not only the presence, but also the relative amounts of specific transcripts being made in cells. Other variant PCR protocols and applications are manifold and often quite inventive! For a list, see Variations on Basic PCR. A recent CRISPR/Cas9 variant amplifies DNA and may lead to PCR at \(37^{\circ}C\), eliminating the need for a thermocycler altogether! This could prove especially useful for conducting PCR in the field. While not yet ready for prime time, check out Cas9 PCR-No Thermocycler! for more. Future technological advances aside, PCR already has broad applications in research, forensic science, history, anthropology, ecological studies of species diversity, and more, and it may even reveal your own genealogy. Let’s look at three of these.

15.5.2.a Forensics

PCR can be applied to identify a person or organism of interest by comparing its DNA to a standard (control) DNA. Figure 15.29 is an example of a polyacrylamide-gel DNA fingerprint.

Using this technology, it is now possible to detect genetic relationships between near and distant relatives (as well as to exclude such relationships), to determine paternity, to demonstrate evolutionary relationships between organisms, and to solve recent and even “cold-case” crimes using DNA left behind on surfaces at crime scenes. Recall that Alu sequences are ~300 bp short-interspersed elements (SINES) that are highly repeated throughout the human genome. DNA fingerprinting is possible in part because each of us has a unique number and distribution of Alu SINEs in our genome. To read more about Alu sequences and human diversity, look at Alu Sequences and Human Diversity. Also see Sir Alec Jeffries to learn about the origins of DNA fingerprinting in real life—and on all those TV CSI programs! For a video on DNA fingerprinting, check out Alu DNA Fingerprinting video.

15.5.2.b History and Society

DNA is a relatively stable molecule (compared to RNA, which you may know is notably unstable!). This property made it the ideal molecular repository of genetic information for all life…, and for DNA fingerprinting. Not only is crime scene DNA stable, In long-dead organisms, DNA can survive in bones and teeth, allowing the identification of Egyptian mummies, the Russian Tsar and his family killed in 1918, and the unearthed English King Richard III.

Using DNA from the living, the missing African American ancestry is emerging, revealing the consequences of slavery in the United States, including its legacy of lingering racism (see Genetic Consequences of American Slave Trade, Micheletti et al., 2020, Amer. J. Human Genet. 107, 265-277). Of more immediate impact on the living is the Innocence Project, started in 1992 in the U.S. The mission of the Project is to use DNA (i.e., DNA fingerprinting) evidence to exonerate persons wrongfully convicted of serious crimes, either on death row or serving long prison terms. As of this writing, the Innocence Project website reports 375 exonerations of mostly males. Of these, 225 (60%) are African American and 29 are LatinX.

15.5.2.c Who Are Your Ancestors?

Tracing your ethnic, racial, and regional ancestry is related to DNA fingerprinting in that it relies on PCR amplification of DNA and comparisons that distinguish markers in large sequence databases. Prices for commercial services have dropped, and their popularity has risen in recent years. You just provide spit or a salivary (buccal) swab. The service amplifies and sequences DNA in your samples and then compares your DNA sequences to database sequences for shared ethnic and regional markers. Based on these comparisons, you are provided with a (more or less) accurate map of your DNA-based ancestry. Folks spending about a hundred dollars (less when on sale!) often ask how accurate these analyses are and what they really mean. For example, what does it mean if your DNA says you are 5% Native American? In fact, different services can sometimes give you different results! Check out DNA Ancestry Testing to get some answers and explanations.

274 The Power of PCR: Some Examples