Figure 8–36 PCR uses repeated rounds of strand separation, hybridization, and synthesis to amplify DNA. As the procedure outlined in Figure 8–35 is repeated, all the newly synthesized fragments serve as templates in their turn. Because the polymerase and the primers remain in the sample after the first cycle, PCR involves simply heating and then cooling the same sample, in the same test tube, again and again. Each cycle doubles the amount of DNA synthesized in the previous cycle, so that within a few cycles, the predominant DNA is identical to the sequence bracketed by and including the two primers in the original template. In the example illustrated here, three cycles of reaction produce 16 DNA chains, 8 of which (boxed in yellow) correspond exactly to one or the other strand of the original bracketed sequence. After four more cycles, 240 of the 256 DNA chains will correspond exactly to the original sequence, and after several more cycles, essentially all of the DNA strands will be this length. Typically, 20–30 cycles are carried out to effectively clone a region of DNA starting from genomic DNA; the rest of the genome remains unamplified, and its concentration is therefore negligible compared with that of the amplified region (Movie 8.2).
subsequent cycles, all the newly synthesized DNA molecules produced by the polymerase serve as templates for the next round of replication (Figure 8–36). Through this iterative amplification process, many copies of the original sequence can be made—billions after about 20 to 30 cycles.
PCR is now the method of choice for cloning relatively short DNA fragments (say, under 10,000 nucleotide pairs). Each cycle takes only about five minutes, and automation of the whole procedure enables cell-free cloning of a DNA fragment in a few hours. The original template for PCR can be either DNA or RNA, so this method can be used to obtain either a genomic clone (complete with introns and exons) or a cDNA copy of an mRNA (Figure 8–37).
The PCR method is extraordinarily sensitive; it can detect a single DNA molecule in a sample if at least part of the sequence of that molecule is known. Trace amounts of RNA can be analyzed in the same way by first transcribing them into DNA with reverse transcriptase. For these reasons, PCR is frequently employed for uses that go beyond simple cloning. For example, it can be used to detect invading pathogens at very early stages of infection. In this case, short sequences complementary to a segment of the infectious agent’s genome are used as primers and following many cycles of amplification, even a few copies of an invading bacterial or viral genome in a patient’s sample can be detected (Figure 8–38). For many infections, PCR has replaced the use of antibodies against microbial molecules to isolate total DNA isolate total mRNA DNA segment to be cloned mRNA sequence to be cloned ADD FIRST PRIMER, REVERSE TRANSCRIPTASE, AND DEOXYRIBONUCLEOSIDE TRIPHOSPHATES SEPARATE STRANDS AND ADD SECOND PRIMER PCR AMPLIFICATION WITH BOTH PRIMERS PRESENTPCR AMPLIFICATION mRNA DNA SEPARATE STRANDS AND ADD PRIMERS genomic clones (A) cDNA clones (B) chromosomal DNA detect the presence of the invader. It is also used to verify the authenticity of a food source—for example, whether a sample of beef actually came from a cow.
Finally, PCR is now widely used in forensics. The method’s extreme sensitivity allows forensic investigators to isolate DNA from minute traces of human blood or other tissue to obtain a DNA fingerprint of the person who left the sample behind.
rare HIV particle control, usingin plasma of blood frominfected person
Figure 8–38 PCR can be used to detect the presence of a viral genome in a sample of blood.