Exponential amplification

The process known as SPREAD (Surface Promoted Replication and Exponential Amplification of DNA Analogues) attempts to reach the target, striven for by many researchers, of an exponential proliferation of biomolecules in model systems. As already mentioned, product inhibition (e.g., by dimerisation of the new matrices to give C2) only allowed parabolic growth. In the SPREAD process, both solid phase chemistry and feeding have a positive effect on the synthesis. Thus, no separation processes are required, as excess reagents can be removed just by washing. The synthetic process consists of four steps ... 

SPREAD surface promoted replication and exponential amplification of DNA analogues... 

Luther, A., Brandsch, R., and von Kiedrowski, G. (1998). Surface promoted replication and exponential amplification. Nature, 396, 245-8. 

The initial opinion about the use of exponential amplifieation strategies for microarray research was sceptical, with many researchers concerned that exponential amplification would introduce too much bias into the analysis (44,45,46,47,48,49,50). There is, however, a growing body of evidence that exponential amplification is indeed a viable option for microarray profiling (33,51,52,53,54,55,56). Exponential amplification strategies can amplify as little as 10 pg of total RNA (roughly equivalent to one cell) while preserving most of the original abundance relationships of the mRNA species (29). 

Subkhankulova T, Livesey FJ. Comparative evaluation of linear and exponential amplification techniques for expression profiling at the single-cell level. Genome Biol 2006 7 R18. 

Figure 6.6. Amplification of DNA by PCR. Target DNA sequence from a complex genome can be amplified by heat denaturation, providing appropriate conditions for the enzyme (Taq DNA polymerase) that allow it to cause exponential amplification of a particular DNA segment. Among components besides the enzyme that are essential for amplification process are oligonucleotide primers in opposite orientation to each other, shown by dotted arrows, deoxynucleotide triphosphates (dNTPs), Mg2+, and buffer. A 30-cycle amplification leads to a many-million-fold amplification of the discrete DNA segment, flanked by oligonucleotide primer sequences. (Reproduced from Short Protocols in Molecular Biology, 4th ed., F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds., Wiley, New York, 1999, p. 15-1.)...

Due to its exponential amplification of target sequences, the extraordinary sensitivity of PCR makes it prone to the amplification of irrelevant sequences if a contaminating DNA sequence exits in the reaction mixture and if the primers are able to prime the contaminating DNA template sequence. Hence extreme precautions are taken to avoid false amplifications due to contamination of template DNA. DNA can be also amplified from RNA by first converting RNA into DNA by an enzyme known as reverse transcriptase hence this method can be used to scan for expression of various genes in different tissues starting with RNA from various tissues. 

In SELEX, multiple rounds of in vitro transcription of random nucleic acid pools, affinity selection, and RT-PCR are performed, thus giving rise to exponential amplification of the selected molecules. The principle underlying SELEX is schematically depicted in Figure 1. After several selection cycles, the binders can subsequently be cloned and sequenced and then characterized. In SELEX, genotype and phenotype are simultaneously represented by the same RNA molecule, since it exerts its function through its three-dimensional structure, which is in turn determined by its nucleotide sequence. The chemical and functional diversity of RNA can be further increased by addition of cofactors such as histidine (Roth and Breaker, 1998) and divalent cations (Tarasow et al, 1997) to the selection. 

Time sequenced, step growth of branch cells and dendrimer structure Self replication of branch cells throughout dendrimer construction Structural proliferation of dendrimers with exponential amplification of branch cells" and surface functionality as a function of generation... 

The cycle is repeated numerous times to achieve exponential amplification of the DNA sequence located between the two primers. 

Luther A, Brandsch R, von Kiedrowski G (1998) Surface-promoted replication and exponential amplification of DNA analogs. Nature 396 245-248... 

It is best to make a large quantity of total RNA from the control cell line (SW620), to be included in every PCR reaction. Typically, the maximum concentrations which allow exponential amplification at 30 cycles for SW620 are cDNA derived from 125 ng input RNA (a 1 8 dilution from the 1 jug RT reaction) for MDR-1 mRNA cDNA derived from 3.91 ng RNA (a 1 256 dilution from the 1 pg RT reaction) for MRP mRNA and cDNA derived from 0.49 ng RNA (a 1 2048 dilution from the 1 pg RT reaction) for p2-microglobulin mRNA. For quantitation of each of these in unknown samples, an initial test PCR reaction is performed using an amount of RNA/cDNA equivalent to that used from SW620. Optimal dilutions of the test sample cDNA for a definitive... 

Two-fold differences in PCR product from 2-fold serial dilutions have been documented for PCR reactions with the primer sets for MDR-1 and MRP shown in Table 1. PCR reactions which do not show two-fold differences from 2-fold serial dilutions may be affected by one of several problems, including those due to PCR reaction components (see Note 6) and those due to competition. Competition limits exponential amplification and accurate quantitation, and can be due to contaminating DNA found in the RNA sample, contaminating PCR products from previous PCR reactions, or amplification of non-specific PCR products. Some samples may have very high levels of MDR-1 mRNA, and therefore competition... 

Another isothermal amplification technique is strand displacement amplification (SDA). " After heat denaturation of DNA in the presence of four primers, dCTP, dGTP dUTP, and a modified deoxynucleotide (dATPaS), two enzymes are added, an exonuclease-deficient polymerase and a restriction enzyme. The two flanking primers that enter into exponential amplification have a restriction site added to their 5 end and get nicked by the restriction enzyme, allow-ing displacement of strands that can in turn be primed, extended, and nicked. Deoxy-ATPotS is used so that the restriction sites include a hemiphosphorothioate linkage to allow single-strand nicking, instead of cutting through double strands. 

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