Biology Reference
In-Depth Information
8.4.13 Random Primers 342
8.4.14 Real-Time PCR 345
8.4.15 Reverse-Transcription PCR
346
8.4.16 TaqMan PCR 346
8.4.17 Digital PCR 346
8.5 Some Research Applications
347
8.5.1
Amplifying Ancient DNA
347
8.5.2
Amplifying Old DNA
350
8.5.3 Amplifying RNA 351
8.5.4
Analysis of mRNA Polyadenylation
352
8.5.5
Cloning a Gene
352
8.5.6
Detecting Gene Amplification
354
8.5.7
Detecting Methylation of DNA
354
8.5.8
Detecting Pathogens in Vector Arthropods
354
8.5.9
Detecting Pesticide Resistance
355
8.5.10 Developmental Biology
356
8.5.11 Engineering DNA 356
8.5.12 Evaluate Efficacy of Disease Control
357
8.5.13 Evolutionary Analyses
357
8.5.14 Sequencing DNA 358
8.6 Multiple Displacement Amplification: Another Method to Amplify DNA
359
8.7 Concluding Remarks
359
References Cited
361
8.1 Overview
Occasionally, a technique is developed that changes the kinds of questions that
can be answered in biology. Within a few short years, the polymerase chain
reaction (PCR) became just such a powerful tool for solving a myriad of basic
and applied problems. Modifications of the PCR continue to be developed and
these permit additional applications.
The PCR is a method for amplifying (copying) small amounts of DNA or RNA.
It can be used to isolate specific DNA fragments, end label DNA, clone cDNA
and genomic DNA, sequence DNA, mutate specific DNA sequences, alter pro-
moters, quantitate the amount of RNA or DNA, and identify molecular markers
for taxonomic or ecological studies. The PCR requires a DNA polymerase, dNTPs,
template DNA, and primers. Information about sequences at each end of the
DNA to be amplified is needed in order to synthesize appropriate primers for
“standard” (allele-specific) PCR. When two specific primers are used, amplifica-
tion of DNA theoretically is geometric, producing large quantities of DNA suit-
able for sequencing, cloning, or probing. PCR methods that use single primers,
such as random amplified polymorphic DNA (RAPD)-PCR, also can result in DNA
