Target Vs Signal Amplification

Nucleic acids can be detected using either target or signal amplification methods. Briefly, target amplification enzymati-cally increases the number of target molecules. In short, "at the end of the day," there are more molecules of the targeted nucleic acid. In contrast, signal amplification does not increase the target but uses highly sensitive reporter molecules or probes to detect the target. For example, in signal amplification, the same number of molecules exists at the end of the day, but the molecular methods act as a kind of "magnifying glass" to aid in their "visualization."

2.1. TARGET AMPLIFICATION Target amplification is the most frequently used method and is accomplished using several technologies. Target amplification increases the amount of the infectious agent's nucleic acid in a test tube by employing an enzymatic in vitro replication step. Examples of target amplification techniques are PCR, transcription-mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), rolling cycle amplification (RCA), ligase chain reaction (LCR), and strand displacement amplification (SDA). All of these

Transcription Mediate Amplification
Fig. 1. Isothermal one dimensional target or probe.

methods use a polymerase or ligase and short synthetic oligonucleotides known as primers. The primers specifically bind to complementary sequences found in the infectious agent. In most of the commercially available kits, an additional level of specificity is added to the assay with a third probe that is complementary to the amplified target sequence. The probe is used to detect the amplicon. The main differences between these approaches are the use of multiple temperatures vs a single temperature and whether the target or probes are linear or circular.

2.2. ISOTHERMAL AMPLIFICATION Isothermal methods acquired their name after the Latin meaning of "iso" being same or similar and after the Greek "thermal (therme)" meaning heat. Isothermal amplification can be further subdivided into methods that target single-dimensional linear nucleic acid targets vs methods that require two-dimensional targets or probes (i.e., circular molecules). Isothermal approaches do not require thermocyclers and is one of the advantages of these methods. In addition, these methods have a high throughput because all steps can be performed in a single tube.

2.2.1. One-Dimensional or Linear Targets/Probes (TMA and NASBA) Figure 1 outlines TMA and NASBA amplification methods (10,11). The most common nucleic acid targets for these techniques are ribosomal RNA (rRNA) sequences that exist in substantial numbers (e.g., exceeding 10,000 copies per organism). Targeting these nucleic acid sequences takes advantage of the naturally occurring multimeric copies of these sequences. To begin, a customized primer with a binding sequence for an RNA polymerase and a region that is complementary to the target rRNA is synthetically made. The region of this primer that is complementary to the rRNA sequence upon hybridization acts as a primer for generating a complementary DNA (cDNA) molecule using a reverse transcriptase enzyme. RNase H activity degrades the rRNA target, leaving the single strand of cDNA. The addition of a DNA polymerase and another complementary DNA primer generates the second strand. The final product results in a double-stranded DNA (dsDNA) copy of the original rRNA sequence plus an RNA polymerase-binding region. The addition of an RNA poly-merase causes this dsDNA to act as a template for the generation of numerous RNA molecules via transcription (hence, the name transcription-mediated amplification). By targeting a high-copy rRNA molecule rather than a DNA sequence that is present at 1-2 copies per organism, every 10,000 rRNAs will be converted to a transcriptional active DNA template capable of producing numerous copies of RNA. Finally, a probe complementary to the amplicon is used to detect the amplified sequence. Although rRNA sequences are most often targeted, this approach can be used for other RNA targets and, with minor adjustments, for DNA targets as well.

2.2.2. Strand Displacement Amplification Like TMA and NASBA, SDA requires multiple enzymes (e.g., a thermostable polymerase and restriction enzyme) (12-14).

Dna Strand DisplacementStrand Displace
Fig. 2. Strand displacement amplification.
Multiple Displacement Amplification
Fig. 3. Rolling circle amplification.

However, in contrast, it requires multiple primers in a specific order (four total) to amplify the target sequence and displace the copied sequence (Fig. 2). An additional difference is its use of a chemically modified deoxynucleotide base (thiolated dCTP). The amplification process uses two phases: the target generation phase and the amplification phase. In the target generation phase, an engineered primer that has a restriction enzyme site incorporated into it binds to its complementary target and initiates strand synthesis using a thermostable poly-merase. A bumper primer displaces the strand generated from the primer containing the restriction enzyme site. Because the newly generated strands incorporate thiolated dCTP, they are not susceptible to restriction enzymatic digestion. A thermostable restriction enzyme introduces a single-strand nick in the double-stranded molecules. The thermostable polymerase then extends the new strand and thereby displaces the strand 3' to the nick. Ultimately, new strands that incorporate this restriction enzyme site lead to the exponential generation of target copies.

2.2.3. Two-Dimensional or Circular Targets/Probes

The RCA mechanism of replication is depicted in Fig. 3. The replication is initiated by a single-stranded nick and begins

Fluorescenee

Fig. 4. (A) Ligase chain reaction; (B) ligase chain reaction detection.

Fluorescenee

Fig. 4. (A) Ligase chain reaction; (B) ligase chain reaction detection.

with the extension of the strand upstream from the nick and proceeds 5' to 3'.The portion of the nicked strand that is 3' or downstream from the nick is displaced as the newly synthesized strand rolls around the circular DNA molecule. The result is a single-stranded tail that forms concatemeric units of the initial circle. Finally, a complementary strand is formed from the single-stranded unit. The rolling circle form of replication is used for the sensitive detection of single-molecule, single-base-point mutations and sequencing applications (15-25). This method has also been useful for whole genomic amplification using high-fidelity and high-processitivity polymerases in the MDA (9). This method uses multiple primers, which bind to the denatured DNA and can generate 33,000 ng of DNA from 0.3 ng of starting material. This method has been shown to replicate the entire genomic sequence in an unbiased fashion. As a result, MDA could prove to be extremely valuable in producing large amounts of rare control material for clinical laboratories.

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Responses

  • austin rosenberg
    What is target amplification?
    2 years ago
  • thomas
    What is target amplification test?
    2 years ago
  • Cecil
    What are the similarities and differences between target amplification and hybridization test?
    2 years ago
  • wilma
    What is targets aplification test?
    2 years ago
  • Uwe
    What is the different between signal amplification and target amplification?
    2 years ago
  • aziza
    What is target amplification in biology?
    11 months ago
  • Mattiesko
    Is molecular signal amplififcation specific?
    6 months ago
  • Teemu Kankkunen
    What is meant by signal amplification provide two examples.?
    4 months ago
  • Fortinbras
    What is signal amplification assay?
    3 months ago
  • catriona stevenson
    What is target amplification technique?
    2 months ago
  • marcho
    Which probe technique is categorized as a target amplification technique?
    2 months ago
  • Carita
    What is signal amplification testing?
    1 month ago
  • Nicol Noakes
    Is signal based amplification a target amplification method?
    1 month ago
  • theodoric
    Which of the following use target amplification methodologies (Choose all that apply.)?
    24 days ago

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