Other methods for the detection of ligase chain reaction products in microtiter

Other methods for the detection of ligase chain

This preview shows page 4 - 5 out of 11 pages.

Other methods for the detection of ligase chain reaction products in microtiter plates are been also used. LCR assays have been developed for the detection of viruses such as HPV, HSV and HIV. Cycling Probe Technology Cycling probe technology is a method for detection and quantification of low amounts of target DNA. The reaction is carried out at a temperature that allows the chimeric probe to anneal to the single-stranded target DNA. RNase H, an enzyme that specifically degrades the RNA portion of the DNA-RNA hybrids, cuts within the RNA portion of the chimeric probe, and the shorter probe fragments dissociate from the target, regenerating the target for further cycling. The resulting accumulation of probe fragments can be detected. Since the target DNA is not amplified, this technique shows low background. Moreover, cycling probe technology is fast, linear, isothermal and simple compared with other DNA detection methods. NEW MOLECULAR TESTS FOR VIROLOGICAL DIAGNOSIS Microarrays A DNA array (or DNA chip) is a collection of spots attached to a solid support where each spot contains one or more single-stranded DNA oligonucleotide fragment [26]. High density arrays, that permit attaching hundred or thousands of oligonucleotides, are referred to as microarrays. A labeled amplification product is hybridized to the probes, and hybridization signals are mapped to several positions within the array. The pattern of hybridization can identify the sequence of PCR, if the number of probes is sufficiently large. The results of hybridization between the bound probe and labeled sequences in the sample applied and tested are revealed by scanning or imaging the array surface. Confocal microscopy is used to scan the chip, detecting fluorescent signals that reveal hybridization at precise locations on the chip. As many DNA sequences can be present on a slide, it is possible for microarray analysis to test for multiple viruses simultaneously. The first application in diagnostic virology has been for rapid sequencing to detect HIV mutations associated with resistance to antiretroviral drugs [27]. Since then, some research groups have developed microarrays that detect several viruses such as respiratory viruses [28], hepatitis C virus [29] and virus causing CNS infection [30]. Multiplexed Microsphere-Based Array Microsphere-based suspension array technologies, such as the Luminex ® xMAP TM system, offer a platform for nucleic acid detection that have some advantages including rapid data acquisition, excellent sensitivity and specificity and multiplexed analysis capability [31]. As compared to planar microarrays, suspension arrays have the advantages of ease of use, low cost, statistical superiority, faster hybridization kinetics and more flexibility in array preparation [31].
Image of page 4
Image of page 5

You've reached the end of your free preview.

Want to read all 11 pages?

  • Fall '12
  • DNA

  • Left Quote Icon

    Student Picture

  • Left Quote Icon

    Student Picture

  • Left Quote Icon

    Student Picture