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Chapter 6:
Detecting Nucleic Acids
Abstract
The polymerase chain reaction has already been described as a biochemical method to copy and detect specific sequences of DNA. PCR or restriction enzymes can also be used as the front end to fragment sizing systems such as gel electrophoresis in order to detect DNA. In this chapter we consider three technologies for detecting DNA: DNA chips, DNA microarrays, and affinity capture. All three of these technologies are based on hybridization of reference strands of DNA to the unknown DNA being tested. DNA chips and DNA microarrays are two-dimensional arrays of reference DNA on glass membranes or microscope slides. Chips are fabricated by synthesizing DNA directly on the substrate. Microarrays are fabricated by printing small volumes of solution containing reference DNA onto the substrate. Affinity capture approaches use beads and other nonplanar surfaces to identify and/or separate hybridized products. Historically, chips used shorter DNA strands than microarrays and affinity capture. These three hybridization technologies may be applied to detection of DNA in a complex environment, such as detecting a pathogen in an environmental sample. The technologies may also be applied to a specific culture to analyze the genetic response to a stimulus by estimating the change in mRNA levels. One significant problem with all DNA array experiments is that the hybridization is not perfect. Errors in hybridization become particularly acute at 90% or greater sequence similarity. Applications that include medical diagnostics and regulatory gene expression studies may require discriminating among very similar subsequences. In these cases, redundant probes specific to unique subsequences may be designed that effectively resequence the region. To put this in perspective, a 1% DNA sequence error rate in the human genome is sufficient to map the sequence from human to chimpanzee.
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CHAPTER 6
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