A Highly Efficient Method for Long-Chain cDNA Synthesis Using Trehalose and Betaine

Spiess, Andrej‐Nikolai; Ivell, Richard

doi:10.1006/abio.2001.5474

Cited by 56 publications

(26 citation statements)

References 21 publications

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“…1B). Additional experiments using the additives betaine, dimethyl sulfoxide, and trehalose did not demonstrate enhanced amplification as others have reported (27,32). High-yield amplification of HCV hemigenomes from acutephase serum.…”

Section: Multikilobase Hcv Rt-pcr Amplicons Obtained From Subjects Wimentioning

confidence: 57%

Accurate Representation of the Hepatitis C Virus Quasispecies in 5.2-Kilobase Amplicons

Netski²,

Mao

et al. 2004

J Clin Microbiol

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Hepatitis C virus (HCV) exists as a swarm of genetically distinct but related variants, or a quasispecies, whose complexity and sequence evolution are critical to studies of viral pathogenesis. Because most studies of the HCV quasispecies have focused on a relatively small genomic segment, the first hypervariable region of the E2 gene, it is possible that viral complexity is occasionally underestimated (due to primer mismatch) and that sequence evolution is misperceived due to unrecognized covariation. This report describes a sensitive and reproducible method to amplify most of the HCV genome as a single 5.2-kb amplicon by using primers directed at relatively conserved genomic segments. Using 52 specimens obtained during acute infection over a range of viral RNA concentrations, the overall rate of successful amplification was 94% and varied in a concentrationdependent manner, with successful amplification in 26 of 26 (100%) specimens at greater than 10 5 IU/ml, 15 of 16 (94%) at 10 4 to 10 5 IU/ml, 6 of 7 (86%) at 10 3 to 10 4 IU/ml, and 2 of 3 (67%) at less than 10 3 IU/ml. Quasispecies complexity, determined by using this novel long-amplicon method followed by heteroduplex mobility assay combined with single-stranded conformational polymorphism (HDA؉SSCP) analysis, was very high, even during acute HCV infection, when 10 to 21 (median, 16) different HDA؉SSCP patterns were detected among 33 cDNA clones examined. Replicate analyses indicate that this diversity is not dominated by random errors generated during amplification. Therefore, the HCV quasispecies is highly complex even during acute infection and is accurately represented in amplicons representing more than half of the viral genome.Hepatitis C virus (HCV) is a medically important human pathogen, infecting about 170 million persons worldwide and representing the single most common cause of liver disease requiring liver transplantation in the United States (2, 3, 34). The viral or host factors that determine the course of HCV infections are unclear, and these studies have been hampered by the lack of a well-characterized small-animal model. Observational studies of infected humans are feasible because of the high prevalence of HCV infection but are limited by small amplicons obtained by conventional reverse transcription-PCR (RT-PCR) methods. Amplification of small genomic segments of HCV by RT-PCR is commonplace and demonstrates the presence of a viral quasispecies or a swarm of genetically related but distinct variants (23).HCV sequence variation is informative, because HCV replicates at high levels while employing its error-prone polymerase and appears to tolerate sequence variation and also because infected individuals apply various selective pressures on the virus. This information will be most valuable if examined longitudinally and comprehensively. Such studies of simian immunodeficiency virus-infected macaques have revealed evidence of immune escape. These studies depended in part on an inbred population of animals. In studies of humans, larger reg...

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Section: Multikilobase Hcv Rt-pcr Amplicons Obtained From Subjects Wimentioning

confidence: 57%

Accurate Representation of the Hepatitis C Virus Quasispecies in 5.2-Kilobase Amplicons

Netski²,

Mao

et al. 2004

J Clin Microbiol

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“…It was also found that trehalose could greatly improve synthesis of long-chain cDNA (8). Our laboratory had previously found that trehalose could improve the overall long PCR performance when used together with several different nanoparticles, such as nanogold (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Aqueous Suspension of Carbon Nanotubes Enhances the Specificity of Long PCR

et al. 2008

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DNA manipulation technology is facing more challenges in the postgenomics era. More and more nanomaterials have been investigated for their potential implications in developing better gene technology. In this study, we reported the beneficial effect of single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) in enhancing the specificity and total efficiency of long (14 kb) PCR. Hydroxylic and carboxylic carbon nanotubes (CNTs) had similar enhancing effects. Nanotubes could become another component for improvements in the amplification of long DNA.

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“…There are also several advanced studies on its intrinsic mechanism, including the concept of replication slippage (Viguera et al, 2001). However, such replication slippage in the HCV 3′UTR cannot be eliminated by existing methods, including the use of polymerases showing strand-displacement activity (Viguera et al, 2001), the alleviation of template structure by reported additives (Spiess et al, 2002; Henke et al, 1997; Hengen et al, 1997; Chou et al, 1992) and the emulation of a crowded reaction environment (Lareu et al, 2007). Only very limited success was achieved in a few freshly-collected HCV serum samples in which single ligation-based RT-PCR has given the expected amplification of the entire HCV 3′UTR (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Enhanced protocol for determining the 3′ terminus of hepatitis C virus

Zhang

Fan

et al. 2010

Journal of Virological Methods

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The determination of viral 3′ ends is a routine practice in molecular biology. However, this has been a challenging task for hepatitis C virus (HCV), an enveloped single-stranded, positive-sense RNA virus classified into the Flaviviridae family. The extreme end of HCV 3′ untranslated region (3′UTR), the so-called 3′ X tail, was not identified at the time of HCV discovery. Complete HCV 3′UTR sequences occupy a very small percentage of the exponentially growing HCV sequence databases. Although commercial kits and experimental protocols are available, these methods are both tedious and not reproducible. A stepwise optimization procedure was developed as a simple and robust protocol for determining the complete HCV 3′UTR from clinical samples. The availability of abundant authentic sequence information for the complete HCV 3′UTR will allow full investigation of its biological role in the life cycle of HCV.

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A Highly Efficient Method for Long-Chain cDNA Synthesis Using Trehalose and Betaine

Cited by 56 publications

References 21 publications

Accurate Representation of the Hepatitis C Virus Quasispecies in 5.2-Kilobase Amplicons

Accurate Representation of the Hepatitis C Virus Quasispecies in 5.2-Kilobase Amplicons

Aqueous Suspension of Carbon Nanotubes Enhances the Specificity of Long PCR

Enhanced protocol for determining the 3′ terminus of hepatitis C virus

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